Compounds, compositions, and methods for modulating ferroptosis and treating excitotoxic disorders

ABSTRACT

The present invention provides, inter alia, a compound having the structure of Formula (I). Also provided are compositions containing a pharmaceutically acceptable carrier and a compound according to the present invention. Further provided are methods for treating or ameliorating the effects of an excitotoxic disorder in a subject, methods of modulating ferroptosis in a subject, methods of reducing reactive oxygen species (ROS) in a cell, and methods for treating or ameliorating the effects of a neurodegenerative disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage Application of InternationalApplication No. PCT/US2014/067977, filed on Dec. 1, 2014, and whichclaims priority to U.S. Provisional Patent Application Nos. 61/910,580,filed on Dec. 2, 2013, and 61/948,242, filed on Mar. 5, 2014. The entirecontents of the above applications are incorporated by reference as ifrecited in full herein.

FIELD OF INVENTION

The present invention provides, inter alia, compounds having thestructure:

Also provided are pharmaceutical compositions containing the compoundsof the present invention, as well as methods of using the compounds andcompositions of the present invention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

This application contains references to amino acids and/or nucleic acidsequences that have been filed concurrently herewith as sequence listingtext file “0365713pct_ST25.txt”, file size of 4.86 KB, created on Nov.24, 2014. The aforementioned sequence listing is hereby incorporated byreference in its entirety pursuant to 37 C.F.R. § 1.52(e)(5).

BACKGROUND OF THE INVENTION

Cell death is crucial for normal development, homeostasis and theprevention of hyper-proliferative diseases such as cancer (Fuchs andSteller, 2011; Thompson, 1995). It was once thought that almost allregulated cell death in mammalian cells resulted from the activation ofcaspase-dependent apoptosis (Fuchs and Steller, 2011; Thompson, 1995).More recently this view has been challenged by the discovery of severalregulated non-apoptotic cell death pathways activated in specificdisease states, including poly(ADP-ribose) polymerase-1 (PARP-1) andapoptosis inducing factor 1 (AIF1)-dependent parthanatos,caspase-1-dependent pyroptosis and receptor interacting protein kinase 1(RIPK1)-dependent necroptosis (Bergsbaken et al., 2009; Christoffersonand Yuan, 2010; Wang et al., 2009). It is believed that additionalregulated forms of non-apoptotic cell death likely remain to bediscovered that mediate cell death in other developmental orpathological circumstances.

The RAS family of small GTPases (HRAS, NRAS and KRAS) are mutated inabout 30% of all cancers (Vigil et al., 2010). Finding compounds thatare selectively lethal to RAS-mutant tumor cells is, therefore, a highpriority. Two structurally unrelated small molecules, named erastin andRSL3, were previously identified. These molecules were selectivelylethal to oncogenic RAS-mutant cell lines, and together, they werereferred to as RAS-selective lethal (RSL) compounds (Dolma et al., 2003;Yang and Stockwell, 2008). Using affinity purification, voltagedependent anion channels 2 and 3 (VDAC2/3) were identified as directtargets of erastin (Yagoda et al., 2007), but not RSL3. ShRNA and cDNAoverexpression studies demonstrated that VDAC2 and VDAC3 are necessary,but not sufficient, for erastin-induced death (Yagoda et al., 2007),indicating that additional unknown targets are required for thisprocess.

The type of cell death activated by the RSLs has been enigmatic. Classicfeatures of apoptosis, such as mitochondrial cytochrome c release,caspase activation and chromatin fragmentation, are not observed inRSL-treated cells (Dolma et al., 2003; Yagoda et al., 2007; Yang andStockwell, 2008). RSL-induced death is, however, associated withincreased levels of intracellular reactive oxygen species (ROS) and isprevented by iron chelation or genetic inhibition of cellular ironuptake (Yagoda et al., 2007; Yang and Stockwell, 2008). In a recentsystematic study of various mechanistically unique lethal compounds, theprevention of cell death by iron chelation was a rare phenomenon (Wolpawet al., 2011), suggesting that few triggers can access iron-dependentlethal mechanisms.

Accordingly, there is a need for the exploration of various pathways ofregulated cell death, as well as for compositions and methods forpreventing the occurrence of regulated cell death. This invention isdirected to meeting these and other needs.

SUMMARY OF THE INVENTION

Without being bound to a particular theory, the inventors hypothesizedthat RSLs, such as erastin, activate a lethal pathway that is differentfrom apoptosis, necrosis and other well-characterized types of regulatedcell death. It was found that erastin-induced death involves a uniqueconstellation of morphological, biochemical and genetic features, whichled to the name “ferroptosis” as a description for this phenotype. Smallmolecule inhibitors of ferroptosis that prevent ferroptosis in cancercells, as well as glutamate-induced cell death in postnatal rat brainslices have been identified and disclosed herein. The inventors havefound an underlying similarity between diverse forms of iron-dependent,non-apoptotic death and that the manipulation of ferroptosis may beexploited to selectively destroy RAS-mutant tumor cells or to preserveneuronal cells exposed to specific oxidative conditions.

Accordingly, one embodiment of the present invention is a compoundaccording to formula (I):

wherein:

-   -   R₁ and R₂, are independently selected from the group consisting        of no atom, H, D, O, halo, C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl, wherein the C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl may be optionally substituted with an        atom or a group selected from the group consisting of halo,        deuterium, C₁₋₄alkyl, CF₃, and combinations thereof; and    -   R₃ is independently selected from the group consisting of H,        C₁₋₁₂aliphatic, C₁₋₆-alkyl-aryl and C₁₋₆-alkyl-heteroaryl;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof,        with the proviso that:    -   when R₃ is ethyl, R₁ and R₂ cannot be both H, O, or

-   -    and    -   when R₃ is ethyl and at least one of R₁ or R₂ is H, R₁ or R₂        cannot be

Another embodiment of the present invention is a compound having theformula:

Another embodiment of the present invention is a pharmaceuticalcomposition. This pharmaceutical composition comprises apharmaceutically acceptable carrier or diluent and a compound accordingto formula (I):

wherein:

-   -   R₁ and R₂, are independently selected from the group consisting        of no atom, H, D, O, halo, C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl, wherein the C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl may be optionally substituted with an        atom or a group selected from the group consisting of halo,        deuterium, C₁₋₄alkyl, CF₃, and combinations thereof; and    -   R₃ is independently selected from the group consisting of H,        C₁₋₁₂aliphatic, C₁₋₆-alkyl-aryl and C₁₋₆-alkyl-heteroaryl;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof,        with the proviso that:    -   when R₃ is ethyl, R₁ and R₂ cannot be both H, O, or

-   -    and    -   when R₃ is ethyl and at least one of R₁ or R₂ is H, R₁ or R₂        cannot be

A further embodiment of the present invention is a kit. This kitcomprises a compound or a pharmaceutical composition according to thepresent invention with instructions for the use of the compound or thepharmaceutical composition, respectively.

Another embodiment of the present invention is a method for treating orameliorating the effects of a disorder in a subject in need thereof.This method comprises administering to the subject an effective amountof a compound having the structure of formula (I):

wherein:

-   -   R₁ and R₂, are independently selected from the group consisting        of no atom, H, D, O, halo, C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl, wherein the C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl may be optionally substituted with an        atom or a group selected from the group consisting of halo,        deuterium, C₁₋₄alkyl, CF₃, and combinations thereof; and    -   R₃ is independently selected from the group consisting of H,        C₁₋₁₂aliphatic, C₁₋₆-alkyl-aryl and C₁₋₆-alkyl-heteroaryl;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof,        with the proviso that:    -   when R₃ is ethyl, R₁ and R₂ cannot be both H, O, or

-   -    and    -   when R₃ is ethyl and at least one of R₁ or R₂ is H, R₁ or R₂        cannot be

An additional embodiment of the present invention is a method fortreating or ameliorating the effects of a disorder in a subject in needthereof. This method comprises administering to the subject an effectiveamount of a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier or diluent and a compound having the structure offormula (I):

wherein:

-   -   R₁ and R₂, are independently selected from the group consisting        of no atom, H, D, O, halo, C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl, wherein the C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl may be optionally substituted with an        atom or a group selected from the group consisting of halo,        deuterium, C₁₋₄alkyl, CF₃, and combinations thereof; and    -   R₃ is independently selected from the group consisting of H,        C₁₋₁₂aliphatic, C₁₋₆-alkyl-aryl and C₁₋₆-alkyl-heteroaryl;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof,        with the proviso that:    -   when R₃ is ethyl, R₁ and R₂ cannot be both H, O, or

-   -    and    -   when R₃ is ethyl and at least one of R₁ or R₂ is H, R₁ or R₂        cannot be

Another embodiment of the present invention is a method of modulatingferroptosis in a subject in need thereof. This method comprisesadministering to the subject an effective amount of a ferroptosisinhibitor, which comprises a compound having the structure of formula(I):

wherein:

-   -   R₁ and R₂, are independently selected from the group consisting        of no atom, H, D, O, halo, C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl, wherein the C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl may be optionally substituted with an        atom or a group selected from the group consisting of halo,        deuterium, C₁₋₄alkyl, CF₃, and combinations thereof; and    -   R₃ is independently selected from the group consisting of H,        C₁₋₁₂aliphatic, C₁₋₆-alkyl-aryl and C₁₋₆-alkyl-heteroaryl;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof,        with the proviso that:    -   when R₃ is ethyl, R₁ and R₂ cannot be both H, O, or

-   -    and    -   when R₃ is ethyl and at least one of R₁ or R₂ is H, R₁ or R₂        cannot be

A further embodiment of the present invention is a method of reducingreactive oxygen species (ROS) in a cell. This method comprisescontacting a cell with a ferroptosis modulator, which comprises acompound having the structure of formula (I):

wherein:

-   -   R₁ and R₂, are independently selected from the group consisting        of no atom, H, D, O, halo, C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl, wherein the C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl may be optionally substituted with an        atom or a group selected from the group consisting of halo,        deuterium, C₁₋₄alkyl, CF₃, and combinations thereof; and    -   R₃ is independently selected from the group consisting of H,        C₁₋₁₂aliphatic, C₁₋₆-alkyl-aryl and C₁₋₆-alkyl-heteroaryl;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof,        with the proviso that:    -   when R₃ is ethyl, R₁ and R₂ cannot be both H, O, or

-   -    and    -   when R₃ is ethyl and at least one of R₁ or R₂ is H, R₁ or R₂        cannot be

An additional embodiment of the present invention is a method fortreating or ameliorating the effects of a neurodegenerative disease in asubject in need thereof. This method comprises administering to thesubject an effective amount of a compound selected from the groupconsisting of

and combinations thereof, or an N-oxide, crystalline form, hydrate, orpharmaceutically acceptable salt thereof.

Another embodiment of the present invention is a compound according toformula (IV):

wherein:

-   -   R₁ is selected from the group consisting of aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle, wherein the aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle may be optionally        substituted with an atom or a group selected from the group        consisting of halo, deuterium, C₁₋₆alkyl, CF₃, and combinations        thereof;    -   R₂ is a triazole, an oxazole, an oxadiazole, or a ketone; and    -   R₃ is a C₃₋₁₂carbocycle, optionally substituted with C₁₋₁₀ alkyl        or halo;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof.

A further embodiment of the present invention is a pharmaceuticalcomposition. This pharmaceutical composition comprises apharmaceutically acceptable carrier or diluent and a compound accordingto formula (IV):

wherein:

-   -   R₁ is selected from the group consisting of aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle, wherein the aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle may be optionally        substituted with an atom or a group selected from the group        consisting of halo, deuterium, C₁₋₆alkyl, CF₃, and combinations        thereof;    -   R₂ is a triazole, an oxazole, an oxadiazole, or a ketone; and    -   R₃ is a C₃₋₁₂carbocycle, optionally substituted with C₁₋₁₀ alkyl        or halo;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof.

An additional embodiment of the present invention is a kit. This kitcomprises a compound or a pharmaceutical composition according to anycompound of the present invention together with instructions for the useof the compound.

Another embodiment of the present invention is a method for treating orameliorating the effects of a disorder in a subject in need thereof.This method comprises administering to the subject an effective amountof a compound having the structure of formula (IV):

wherein:

-   -   R₁ is selected from the group consisting of aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle, wherein the aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle may be optionally        substituted with an atom or a group selected from the group        consisting of halo, deuterium, C₁₋₆alkyl, CF₃, and combinations        thereof;    -   R₂ is a triazole, an oxazole, an oxadiazole, or a ketone; and    -   R₃ is a C₃₋₁₂carbocycle, optionally substituted with C₁₋₁₀ alkyl        or halo;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof.

A further embodiment of the present invention is a method for treatingor ameliorating the effects of a disorder in a subject in need thereof.This method comprises administering to the subject an effective amountof a pharmaceutical composition comprising a pharmaceutically acceptablecarrier or diluent and a compound having the structure of formula (IV):

wherein:

-   -   R₁ is selected from the group consisting of aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle, wherein the aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle may be optionally        substituted with an atom or a group selected from the group        consisting of halo, deuterium, C₁₋₆alkyl, CF₃, and combinations        thereof;    -   R₂ is a triazole, an oxazole, an oxadiazole, or a ketone; and    -   R₃ is a C₃₋₁₂carbocycle, optionally substituted with C₁₋₁₀ alkyl        or halo;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof.

Another embodiment of the present invention is a method of modulatingferroptosis in a subject in need thereof. This method comprisesadministering to the subject an effective amount of a ferroptosisinhibitor, which comprises a compound having the structure of formula(IV):

wherein:

-   -   R₁ is selected from the group consisting of aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle, wherein the aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle may be optionally        substituted with an atom or a group selected from the group        consisting of halo, deuterium, C₁₋₆alkyl, CF₃, and combinations        thereof;    -   R₂ is a triazole, an oxazole, an oxadiazole, or a ketone; and    -   R₃ is a C₃₋₁₂carbocycle, optionally substituted with C₁₋₁₀ alkyl        or halo;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof.

An additional embodiment of the present invention is a method ofreducing reactive oxygen species (ROS) in a cell. This method comprisescontacting a cell with a ferroptosis modulator, which comprises acompound having the structure of formula (IV):

wherein:

-   -   R₁ is selected from the group consisting of aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle, wherein the aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle may be optionally        substituted with an atom or a group selected from the group        consisting of halo, deuterium, C₁₋₆alkyl, CF₃, and combinations        thereof;    -   R₂ is a triazole, an oxazole, an oxadiazole, or a ketone; and    -   R₃ is a C₃₋₁₂carbocycle, optionally substituted with C₁₋₁₀ alkyl        or halo;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof.

Another embodiment of the present invention is a compound having theformula:

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F show that erastin-induced death triggers the accumulation ofcytosolic ROS whose production can be inhibited by the iron chelatordeferoxamine (DFO). FIG. 1A shows representative microscopy images ofHT-1080 cell viability over time +/−erastin (Era, 10 μM) anddeferoxamine (DFO, 100 μM). FIGS. 1B and 1C show cytosolic and lipid ROSproduction assessed over time (2, 4 and 6 hours) by flow cytometry usingH₂DCFDA and C11-BODIPY. FIG. 1D shows mitochondrial ROS assessed inHT-1080 cells treated for 6 hours with erastin +/−DFO, as above, or withrotenone (250 nM)+/−DFO. In FIGS. 1A-D, representative data from one offour experiments are shown. FIG. 1E shows erastin-induced death in 143Bρ⁰ and ρ⁺ cells. FIG. 1F shows mtDNA-encoded transcript levels in ρ⁰ andρ⁺ cells. Results in FIGS. 1E and 1F are mean±SD from one of threerepresentative experiments.

FIGS. 2A-2E show that erastin-induced oxidative death is iron-dependent.FIG. 2A shows a transmission electron microscopy image of BJeLR cellstreated with DMSO (10 hours), erastin (37 μM, 10 hours), staurosporine(STS, 0.75 μM, 8 hours), H₂O₂ (16 mM, 1 hour) and rapamycin (Rap, 100nM, 24 hours). Single white arrowheads show shrunken mitochondria,paired white arrowheads show chromatin condensation, black arrowheadsshow cytoplasmic and organelle swelling, plasma membrane rupture, andblack arrow shows formation of double-membrane vesicles. A minimum of 10cells per treatment condition were examined. FIG. 2B shows normalizedATP levels in HT-1080 and BJeLR cells treated as in FIG. 2A with theindicated compounds. Representative data (mean±SD) from one of threeindependent experiments are shown. FIG. 2C shows modulatory profiling ofknown small molecule cell death inhibitors in HT-1080, BJ-eLR and Calu-1cells treated with erastin (10 μM, 24 hours). FIG. 2D shows the effectof inhibitors on H₂DCFDA-sensitive ROS production in HT-1080 cellstreated for 4 hours. FIG. 2E shows modulatory profiling of ciclopiroxolamine (CPX), DFO, ebselen (Ebs), trolox (Tlx), U0126 and CHX onoxidative and non-oxidative lethal agents.

FIGS. 3A-3G show that erastin-induced ferroptosis exhibits a uniquegenetic profile. FIG. 3A shows an outline of the MitoCarta shRNA screenand confirmation pipeline. FIGS. 3B and 3C show six high confidencegenes required for erastin-induced ferroptosis. FIG. 3B shows theviability of HT-1080 cells infected with shRNAs for 72 hours and treatedwith erastin (10 μM, 24 hours). FIG. 3C shows the mRNA levels forhairpins shown in FIG. 3B determined using RT-qPCR. Data in FIGS. 3B and3C are mean±SD from one of three experiments. The sequences of variousclones of shRNA listed in FIGS. 3B and 3C are as follows: the sequencefor sh263-VDAC3 is shown in SEQ ID NO:1, the sequence for sh548-RPL8 isshown in SEQ ID NO:2, the sequence for sh440-ATP5G3 is shown in SEQ IDNO:3, the sequence for sh978-TTC35 is shown in SEQ ID NO:4, the sequencefor sh2326-IREB2 is shown in SEQ ID NO:5, the sequence for sh1776-ACSF2is shown in SEQ ID NO:6, and the sequence for sh332-CS is shown in SEQID NO:7. FIGS. 3D and 3E show the effect of shRNA-mediated silencing ofhigh-confidence genes using the best hairpin identified by mRNAsilencing efficiency in FIG. 3C on cell viability. FIG. 3D shows theviability of various cell lines treated with a lethal dose of erastin(indicated in parentheses) for 24 hours. FIG. 3E shows the viability ofHT-1080 cells treated with various death-inducing or cytostaticcompounds. For FIGS. 3D and 3E, % rescue was computed relative to eachshRNA alone+DMSO. FIG. 3F is a cartoon outline of glutamine (Gln)metabolism. Shaded box indicates mitochondria. FIG. 3G shows images ofHT-1080 cells treated with aminooxyacetic acid (AOA)+/−dimethylalphaketoglutarate (DMK)+/−erastin.

FIGS. 4A-4K show the identification and characterization ofFerrostatin-1. FIG. 4A shows the structure of ferrostatin-1 (Fer-1).FIG. 4B shows the effect of resynthesized Fer-1 (0.5 μM) on thelethality of various compounds in HT-1080 cells. FIG. 4C shows theeffect of Fer-1 and U0126 on ERK phosphorylation in HT-1080 cells. FIG.4D shows the effect of DFO, CHX, trolox (Tlx) and Fer-1 on HT-1080 cellproliferation over 48 hours as assessed by Vi-Cell. FIG. 4E shows theeffect of Fer-1 (0.5 μM) on erastin (10 μM)-induced ROS production inHT-1080 cells (4 hour treatment). FIG. 4F shows cell-free antioxidantpotential monitored by changes in the absorbance at 517 nm of the stableradical DPPH. FIG. 4G shows the dose-response relationship forinhibition of erastin (10 μM, 24 hours)-induced death in HT-1080 cellsby Fer-1 and analogs. FIG. 4H shows the structure of various compoundslisted in FIG. 4G. FIG. 4I shows the correlation between predictedpartition coefficient (log P) and the ability of various Fer-1 analogsto prevent erastin-induced death. FIG. 4J shows the dose-responserelationship for inhibition of erastin (10 μM, 24 hours)-induced deathby various antioxidants. FIG. 4K shows a plot of predicted partitioncoefficient (log P) and ability of various antioxidants to preventerastin-induced death. Data in FIGS. 4B, 4D, 4F, 4G, and 4J representmean±SD from one of three representative experiments.

FIGS. 5A-5E show the effects of Fer-1 on excitotoxic cell death inorganotypic hippocampal slice cultures. FIG. 5A is a cartoon outline ofthe hippocampal slice procedure used herein. FIG. 5B shows bright-fieldand fluorescent images of propidium iodide (PI) staining of treatedhippocampal slices. Slices were treated with glutamate (5 mM, 3hours)+/−Fer-1 (2 μM), CPX (5 μM) or MK-801 (10 μM). Representativeimages from 1 one 6 slices per condition are shown. FIGS. 5C-E showquantification of the effects depicted in FIG. 5B. Data were analyzedusing a two-way ANOVA (brain region×drug treatment) followed byBonferroni post-tests. *: P<0.05, **: P<0.01, ***: P<0.001.

FIGS. 6A-6I show that erastin inhibits the activity of system x_(c) ⁻.FIG. 6A shows the modulatory profile of HT-1080 cells treated withdifferent lethal compounds and inhibitors. FIG. 6B is a cartoondepicting the composition and function of system L and system x_(c) ⁻.Cys: cystine, NAA: neutral amino acids. FIG. 6C shows SLC7A11 mRNAlevels in compound (6 hours)-treated HT-1080 cells determined byRT-qPCR. FIGS. 6D and 6E show the effect of silencing SLC7A11 usingsiRNA on erastin (10 μM, 8 hours)-induced death (FIG. 6D) and mRNAlevels (FIG. 6E) in HT-1080 cells. FIG. 6F shows Na⁺-independent[¹⁴C]cystine uptake by HT-1080 cells in response to various drugs. FIG.6G shows identification of SLC7A5 as the lone target identified byerastin affinity purification in both BJeH and BJeLR cells. FIG. 6Hshows the metabolic profiling of system L and non-system L substrateamino acid levels in erastin-treated Jurkat cells. FIG. 6I shows theeffect of L-glutamic acid (L-Glu, 12.5 mM) and D-phenylalanine (D-Phe,12.5 mM) on erastin-induced death in HT-1080 cells.

FIGS. 7A-7E show the role of NOX in erastin-induced death. FIG. 7A showsthe outline of the NOX (NADPH oxidase) pathway. Inhibitors are shown ingray. FIG. 7B shows the effect of NOX pathway inhibitors onerastin-induced death in Calu-1 and HT-1080 cells. GKT: GKT137831. FIGS.7C and 7D show the effect of shRNA silencing of the PPP enzymesglucose-6-phosphate dehydrogenase (G6PD) and phosphogluconatedehydrogenase (PGD) on viability of erastin (2.5 μM)-treated Calu-1cells. Infection with shRNA targeting VDAC2 was used as a positivecontrol. Relative mRNA levels in (FIG. 7D) were assessed by qPCRfollowing shRNA knockdown. Data in FIGS. 7B, 7C, and 7D representsmean±SD. FIG. 7E shows a model of ferroptosis pathway. The coreferroptotic lethal mechanism is in the shaded portion.

FIGS. 8A-8E show that RSLs trigger iron-dependent cell death independentof the mitochondrial electron transport chain. FIG. 8A shows theviability of HT-1080 cells treated with erastin +/−ferric ammoniumcitrate (FAC) as assessed in triplicate by Vi-Cell. FIG. 8B shows theviability of HT-1080 cells treated with DMSO or erastin +/−FAC, ferriccitrate (FC), iron chloride hexahydrate (IHC), manganese chloride (Mn),nickel sulfate hexahydrate (Ni), cobalt chloride hexahydrate (Co) orcopper sulfate (Cu). FAC was used at 10 μg/mL, all other metals wereused at 25 μM. Cell viability was assessed by Trypan Blue exclusion(Vi-Cell) in triplicate and the effects of the erastin+metal combinationwere expressed as a percentage of the DMSO+metal viability alone. FIGS.8C and 8D show mitochondrial superoxide levels in 143B cells assessed byflow cytometry using MitoSOX. Treatments used: 250 nM rotenone (Rote),100 μM DFO alone or in combination, as indicated. FIG. 8E shows theviability of 143B ρ⁺ and ρ⁰ cells treated for 24 hours with RSL3 andassessed by Alamar Blue. All experiments were repeated two to four timeswith similar results, and representative data from one experiment areshown. Data in FIGS. 8A, 8B and 8E represent mean±SD from multiplereplicates within one experiment.

FIGS. 9A-9C show that ferroptosis occurs in mouse embryonic fibroblasts(MEFs), independent of Bax and Bak, and can be attenuated by the lateaddition of inhibitors. FIG. 9A shows the viability of SV40-transformedMEFs (control and Bax/Bak double knockout, DKO) that were treated witherastin +/−DFO, Trolox, U0126 or cycloheximide (CHX) for 24 hours asindicated. FIG. 9B shows the viability of wild-type and DKO MEFs thatwere treated with staurosporine (STS) for 24 hours at the indicatedconcentrations to induce apoptosis. Bax/Bak double knockout MEFs aremore resistant to STS, as expected. In FIGS. 9A and 9B, cell viabilitywas assessed by Alamar Blue. Experiments were repeated twice withsimilar results and representative data from one experiment are shown.All values are mean±SD from multiple replicates within each experiment.FIG. 9C shows microscopy images of cells that were treated +/−erastinand co-treated with the indicated inhibitors. Inhibitors were addedeither at the same time as erastin (0 hours) or 2-6 hours later (+2, +4,+6 hours). 2,2-bipyridyl (2,2-BP) is a membrane permeable iron chelator.All cells were photographed 24 hours after the start of the experiment.This experiment was repeated three times with similar results, andrepresentative data from one experiment is shown.

FIGS. 10A-10D show various methods of validating the role of IREB2 inferroptosis. In FIGS. 10A-10C, HT-1080 cells were infected with shRNAstargeting IREB2 and FBXL5 for 3 days and then examined for geneexpression or drug sensitivity. FIGS. 10A and 10B show reciprocaltranscriptional regulation of iron-regulated genes induced by silencingof IREB2 and FBXL5 as assessed by RT-qPCR. DFO treatment (48 hours) wasused as a control for changes in gene expression. ISCU, FTH1, FTL andTFRC are known iron-regulated genes (Sanchez et al., 2011). FIG. 10Cshows silencing of FBXL5 sensitizes cells to erastin-induced death. FIG.10D shows that aminooxyacetic acid (AOA), but not dichloroacetic acid(DCA), inhibits erastin-induced death in HT-1080 and BJeLR cells. Alldata are mean±SD from multiple replicates within one experiment. Allexperiments were performed 2-4 times with similar results.Representative data from one experiment are shown.

FIGS. 11A-11B show Fer-1, and a Fer-1 structure-activity relationship(SAR) analysis. FIG. 11A shows that the re-testing in 10-point, 2-folddilution series of the top four compounds from a LOC (Lead OptimizedCompound) library screen validated to suppress erastin-induced death inHT-1080 cells. FIG. 11B shows the structure of these top 4 compounds.

FIGS. 12A-12E show the analysis of the role of calcium and system x_(c)⁻ in ferroptosis. FIG. 12A shows the viability of HT-1080 cells treatedfor 24 hours with erastin +/−DMSO, the calcium chelators BAPTA-AM orFura-2, or, as a positive control for death rescue, the iron chelatorciclopirox olamine (CPX), at the indicated concentration. FIG. 12B showsthe viability of HT-1080 cells treated for 24 hours with erastin,monosodium L-glutamic acid or RSL3+/−inhibitors using Alamar Blue. FIG.12C shows that sulfasalazine, like erastin, displays RAS-selectivelethal properties in the BJ cell series assay. For FIGS. 12A-12C, valuesrepresent mean±SD from multiple replicates within one experiment. Theentire experiment was repeated twice and representative data from oneexperiment are shown. FIG. 12D shows microscopy images of HT-1080 cellsthat were transfected for 48 hours with either a control plasmid(pMaxGFP) or pCMV6-SLC7A11-DDK then treated with DMSO, erastin or SAS,as indicated, and photographed. FIG. 12E shows [¹⁴C]-cystine uptake intoHT-1080 cells measured under Na⁺-free conditions in response to DMSO,erastin and RSL3. Data represents mean±SD, n=3.

FIGS. 13A-13B show that erastin-induced death is prevented by inhibitionof the PPP/NOX pathway. FIG. 13A shows relative expression of NOX familycatalytic subunit mRNAs in Calu-1 cells assessed by RT-qPCR. FIG. 13Bshows the viability of Calu-1 and BJeLR cells in response to erastin (10μM)+/−the PPP inhibitor 6-aminonicotinamde (6-AN, 200 μM) after 24 hoursby Vi-Cell. Data in FIGS. 13A and 13B represent mean±SD of replicatesfrom one experiment. FIG. 13C shows H₂DCFDA-reactive ROS measured inBJeLR cells that were treated for 8.5 hours, as indicated, prior to theonset of overt death in these cells. Experiments were performed at 3times with similar results, and representative data from one experimentare shown.

FIGS. 14A-14D show the results of mouse microsomal stability testing forcertain Fer-1 analogs: SRS15-72 (FIG. 14A), SRS16-80 (FIG. 14B),SRS16-96 (FIG. 14C), and a control (FIG. 14D).

FIGS. 15A-C show the effects of ferrostatins in Huntington's disease(HD). FIG. 15A shows the effect of ferrostatins on cell survival in anHD brain-slice model. YFP=yellow fluorescent protein transfectioncontrol. httN90Q73 is mutant huntingtin (N-terminal 90aa with a Q73repeat). KW+SP is a combination used as a positive control forprotection. FIG. 15B shows a dose-response test of the effect offerrostatins in a model of PVL. Cys=Cystine supplementation, a positivecontrol for cell death rescue. FIG. 15C shows the effect offerrostatins, at various concentrations, in a primary kidney renaltubule damage model.

FIGS. 16A-C show that Fer-1 does not inhibit all forms of ROS productionor ROS-inducted death. FIG. 16A shows mitochondrial ROS production inresponse to rotenone (Rot, 250 nM, 3 hr)+/−Fer-1 (1 μM) was detectedusing MitoSOX. FIG. 16B shows cardiolipin peroxidation in response tostaurosporine (STS, 100 nM, 3 hr) detected using 10-nonyl acridineorange (NAO). Data in FIGS. 16A-B were analyzed by one-way ANOVA ***P<0.001, ns=not significant. FIG. 16C shows lysosomal membranepermeabilization detected in response to H₂O₂ using acridine orange (AO)re-localization. An iron chelator, ciclopirox olamine (CPX), protectsfrom lysosomal rupture.

FIG. 17 shows an SAR of Fer-1 based on potency in the HT-1080death-suppression.

FIGS. 18A-B show an SAR study of Fer-1. FIG. 18A shows EC50, cLogP, and% DPPH inhibition of selected potent ferrostatins. DPPH:2,2-diphenyl-1-picrylhydrazyl radical. FIG. 18B shows an X-ray structureof the most potent Fer-1 analog (SRS11-92).

FIG. 19 shows Fer-1 analogs with sterically shielding aminesubstituents.

FIG. 20 shows Fer-1 analogs incorporating ester bioisosteres.

FIG. 21 shows that Fer-1 and SRS16-86 suppress cell death in an in vivokidney ischemia reperfusion injury mode (IRI). Mice were dosed IP with 2mg/kg Fer-1 or SRS16-86, or vehicle 30 minutes prior to surgery toinduce IRI. Serum creatinine is used as a marker of kidney damage.

FIG. 22 shows the generation of doxycyclin-inducible kidney tubularspecific conditional FADD- and caspase-8 knockout mice. (A-D)Representative genotyping of either FADD-inducible or caspase-8doxycyclin-inducible kidney tubular-specific conditional knockout mice.(E) Indistinguishability of fl/wt and fl/fl mice after doxycyclintreatment for 3 wk via the drinking water (see Materials and Methods fordetails). (F) Mice of the indicated genotypes were treated for 0 wk, 1wk, 2 wk, or 3 wk with doxycyclin before detection of serum lipase(Upper) and serum urea (Lower) levels (n=3-8 per group for all bloodvalues).

FIG. 23 shows that conditional deletion of FADD or caspase-8 does notsensitize renal tubules to necroptosis. (A) Scheme of thedoxycyclin-inducible conditional tubular knockout. (B) After 3 weeks ofdoxycycline treatment, no detection of the FADD protein in freshlyisolated renal tubules was possible; L929 cells serve as a positivecontrol. (C) Periodic acid-Schiff (PAS) staining of normal renalmorphology in Pax8-rtA; Tet-on.Cre×FADD fl/fl mice after 3-wk treatmentwith doxycyclin via the drinking water. (D-F) Similarly, caspase-8 wasinducibly depleted from tubules. Note that the anti-mouse monoclonalantibody against caspase-8 cross-reacts with a nonspecific protein justbelow the band of caspase-8. Mouse embryonic fibroblasts (MEFs) fromcaspase-8/RIPK3-dko mice serve as a negative control, MEFs fromcyclophilin D-deficient ppif-ko mice serve as a positive control, as dothe C57BL/6 WT mice. (G) Serum creatinine levels remain in the normalrange in all mice investigated as indicated. (H) Doxycyclin-inducedconditional FADD-deficient or caspase-8-deficient mice react to 20 mg/kgbody weight cisplatin-induced acute kidney injury similarly tononstimulated mice. (I) Necrostatin-1 (Nec-1; 50 μM) does not influencethe amount of LDH released from hypoxic renal tubules, either in thepresence (Left) or absence (Right) of glycine (glc) (n=8-10 per group).

FIG. 24 shows preparation of murine renal tubules for Western blotanalysis. (A) Classical appearance after performance of the initialenzymatic preparation steps (see Example 17; Materials and Methods fordetails). (B) Overview of hand-picked tubular segments. For Western blotanalysis, proximal tubules were collected due to the very highsusceptibility to undergo necrosis in acute kidney injury. (C)Doxycyclin-fed mice were killed after 3 wk of transactivator induction,and renal tubules were collected. Proximal tubular segments weresubsequently pooled and loaded onto gels for Western blotting in FIG.23. DCT, distal convoluted tubule; TAL, thick ascending limb.

FIG. 25 shows the phenotype of RIPK3-deficient mice. (A) Example ofhigh-resolution intravital microscopy. Green, autofluorescence of thekidney tubules; red, 70 kDa rhodamine dextran (applied via a jugularcatheter); yellow, Texas Red (2 kDa) dye, which is freely filtered inthe glomerular and reabsorbed in the proximal tubule, counterstainingthe endolysosomal compartment; PC, peritubular capillary. (B and C)Intravital microscopy reveals significantly increased peritubulardiameters in untreated RIPK3-deficient mice compared with WTlittermates. Data in C were generated by counting the diameters ofperitubular capillaries at a number of at least 1.115 per group. (D)Based on the data presented in B, the increase in peritubular diametersis associated with 125.7% overall organ perfusion in RIPK3-deficientmice. (E) Model of cerulein-induced pancreatitis (CIP) in WT andRIPK3-deficient mice. No significant protection from increased serumconcentrations of serum lipase or serum amylase was detected inRIPK3-deficient mice compared with WT mice (n=7 mice per group). (F andG) Failure to gain overall body weight in both female (Left) and male(Right) RIPK3-deficient mice compared with corresponding littermates,followed for 30 wk after an age of 4 wk (n=8-21 per group).

FIG. 26 shows that ferroptosis mediates synchronized tubular necrosisand contributes to immune-cell extravasation into ischemic tissue. (A)Snapshots of functional freshly isolated proximal renal tubule segmentsundergoing rapid synchronized necrosis upon fatty-acid depletion. (B) Inthe presence of fatty acids, addition of erastin accelerated, whereasSRS16-86 (a third-generation ferrostatin; see FIG. 27) prevented,tubular necrosis. (C) During the time course ofhydroxyquinoline/Fe-induced tubular necrosis, Fer-1 and theNox-inhibitor GKT prevented LDH release whereas Nec-1 did not showprotection. (D) Reflected light oblique transillumination imaging ofleukocyte passages through postcapillary venules in IRI of the cremastermuscle in the presence of vehicle or Fer-1. (E) FITC dextran leakage inthe same model. (F) Leukocyte rolling is affected by Fer-1 within thefirst hour after reperfusion. (G) Significantly reduced leukocytetransmigration in the presence of Fer-1.

FIG. 27 shows generation and in vitro testing of a ferrostatinderivative for in vivo applications. (A) Synthetic route of the mostmicrosomal and plasma stable ferrostatin analog (SRS16-86). (B) Modelstructure of the two novel ferrostatin derivates SRS16-79 (inactivecompound) and SRS16-86 (active compound). (C) FACS analysis for thenecrotic marker 7AAD and phosphatidylserine accessibility (annexin V) inHT1080 and NIH 3T3 cells treated with either vehicle or 50 μM erastin inthe presence or absence of 1 μM Fer-1, SRS16-86, or SRS16-79. (D)Absence of cleaved caspase-3 in necroptosis and ferroptosis. Westernblot of cleaved caspase-3 in lysates from 100 ng/mL TNFα plus 1 μM Smacmimetics plus 25 μM zVAD-treated HT-29 cells, 50 μM erastin-treated NIH3T3 cells, and 50 μM erastin-treated HT1080 cells for 24 h. Monoclonalanti-Fas-treated Jurkat cells (100 ng/mL) served as positive control.TSZ, TNFa/SMAC-mimetic/zVAD.

FIG. 28 shows that leukocyte adherence remains unchanged in IRI of thecremaster muscle upon addition of Fer-1. Intravital evaluation ofintravascular firm adhesion of leukocytes in postcapillary venulesfollowing 1 h and 2 h after onset of reperfusion.

FIG. 29 shows that ferroptosis contributes to crystal-induced acutekidney injury, but not to the LPS-induced shock model. (A) PAS andPizzolato staining of mice treated with CaOx and Fer-1. (B) CaOxdeposition is not different between the two groups. (C) Evaluation offunctional markers of acute kidney injury in mice that underwentCaOx-induced nephropathy. (D) Fer-1 significantly reduces theultrastructural damage in CaOx nephropathy. (E) Significant reduction ofexpression of Kim-1 (TIM-1), IL-6, CXCL₂, and p65 in the Fer-1-treatedCa-Ox model (*P=0.05-0.02, **P=0.02-0.001, ***P≤0.001; n=8-10 pergroup). (F and G) In the model of LPS-induced septic shock, 12 mice pergroup were injected intraperitoneally with LPS as described in Example17, Materials and Methods. (F) Temperature drop and (G) overall survivalrates following 96 h after LPS injection (n=12 per group, error barsindicate SEM).

FIG. 30 shows that ferroptosis significantly contributes to renalischemia-reperfusion injury. (A and B) Representative PAS stainings andquantification of renal damage from mice treated with severe ischemicdurations before reperfusion. Mice were killed 48 h after reperfusion.(C and D) Functional markers of acute kidney injury after severe IRI (asin A and B). (E and F) Histologic PAS staining and quantification ofsham operation or ultrasevere IRI (50 min of ischemia beforereperfusion) in WT mice treated with (Nec-1+SfA) combination therapytogether with either SRS16-79 or SRS16-86. (G and H) C57BL/6 weretreated as in E, and functional markers of acute kidney injury weremeasured 48 h after the onset of reperfusion. n.s., not significant;*P=0.05-0.02, **P=0.02-0.001, ***P≤0.001; n=10 per group in allexperiments).

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, new analogs of Fer-1 are provided. Certain ofthe analogs have improved microsomal stability and solubility whilestill maintaining good inhibition potency of ferroptosis. Accordingly,one embodiment of the present invention is a compound according toformula (I):

wherein:

-   -   R₁ and R₂, are independently selected from the group consisting        of no atom, H, D, O, halo, C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl, wherein the C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl may be optionally substituted with an        atom or a group selected from the group consisting of halo,        deuterium, C₁₋₄alkyl, CF₃, and combinations thereof; and    -   R₃ is independently selected from the group consisting of H,        C₁₋₁₂aliphatic, C₁₋₆-alkyl-aryl and C₁₋₆-alkyl-heteroaryl;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof,        with the proviso that:    -   when R₃ is ethyl, R₁ and R₂ cannot be both H, O, or

-   -    and    -   when R₃ is ethyl and at least one of R₁ or R₂ is H, R₁ or R₂        cannot be

In one aspect of this embodiment, the compound has the structure offormula (II):

wherein:

-   -   R₃ is independently selected from the group consisting of H,        C₁₋₁₂aliphatic, C₁₋₆-alkyl-aryl and C₁₋₆-alkyl-heteroaryl;    -   R₄ and R₅ are independently selected from the group consisting        of no atom, H, D, O, halo, aryl, heteroaryl, C₁₋₆alkyl,        C₁₋₆alkyl-aryl, and C₁₋₆alkyl-heteroaryl; and    -   is an optional bond,        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof.

In another aspect of this embodiment, the compound has the structure offormula (III):

wherein the C₂₋₄ alkyl is selected from the group consisting of ethyl,isopropyl, n-propyl, butyl, and t-butyl,or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptablesalt thereof.

In another aspect of this embodiment, the compound is selected from thegroup consisting of:

or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptablesalt thereof.

Preferably, the compound is selected from the group consisting of:

and combinations thereof, or an N-oxide, crystalline form, hydrate, orpharmaceutically acceptable salt thereof.

Another embodiment of the present invention is a compound having theformula:

Another embodiment of the present invention is a pharmaceuticalcomposition. This pharmaceutical composition comprises apharmaceutically acceptable carrier or diluent and a compound accordingto formula (I):

wherein:

-   -   R₁ and R₂, are independently selected from the group consisting        of no atom, H, D, O, halo, C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl, wherein the C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl may be optionally substituted with an        atom or a group selected from the group consisting of halo,        deuterium, C₁₋₄alkyl, CF₃, and combinations thereof; and    -   R₃ is independently selected from the group consisting of H,        C₁₋₁₂aliphatic, C₁₋₆-alkyl-aryl and C₁₋₆-alkyl-heteroaryl;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof,        with the proviso that:    -   when R₃ is ethyl, R₁ and R₂ cannot be both H, O, or

-   -    and    -   when R₃ is ethyl and at least one of R₁ or R₂ is H, R₁ or R₂        cannot be

Suitable and preferred compounds that are used in the pharmaceuticalcompositions of the present invention are disclosed above in formulas(I)-(III), including the particular SRS compounds also identified above.

A further embodiment of the present invention is a kit. This kitcomprises a compound or a pharmaceutical composition disclosed hereinwith instructions for the use of the compound or the pharmaceuticalcomposition, respectively.

The kits may also include suitable storage containers, e.g., ampules,vials, tubes, etc., for each compound of the present invention (which,e.g., may be in the form of pharmaceutical compositions) and otherreagents, e.g., buffers, balanced salt solutions, etc., for use inadministering the active agents to subjects. The compounds and/orpharmaceutical compositions of the invention and other reagents may bepresent in the kits in any convenient form, such as, e.g., in a solutionor in a powder form. The kits may further include a packaging container,optionally having one or more partitions for housing the compoundsand/or pharmaceutical compositions and other optional reagents.

Another embodiment of the present invention is a method for treating orameliorating the effects of a disorder in a subject in need thereof.This method comprises administering to the subject an effective amountof a compound having the structure of formula (I):

wherein:

-   -   R₁ and R₂, are independently selected from the group consisting        of no atom, H, D, O, halo, C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl, wherein the C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl may be optionally substituted with an        atom or a group selected from the group consisting of halo,        deuterium, C₁₋₄alkyl, CF₃, and combinations thereof; and    -   R₃ is independently selected from the group consisting of H,        C₁₋₁₂aliphatic, C₁₋₆-alkyl-aryl and C₁₋₆-alkyl-heteroaryl;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof,        with the proviso that:    -   when R₃ is ethyl, R₁ and R₂ cannot be both H, O, or

-   -    and    -   when R₃ is ethyl and at least one of R₁ or R₂ is H, R₁ or R₂        cannot be

As used herein, the terms “treat,” “treating,” “treatment” andgrammatical variations thereof mean subjecting an individual subject toa protocol, regimen, process or remedy, in which it is desired to obtaina physiologic response or outcome in that subject, e.g., a patient. Inparticular, the methods and compositions of the present invention may beused to slow the development of disease symptoms or delay the onset ofthe disease or condition, or halt the progression of diseasedevelopment. However, because every treated subject may not respond to aparticular treatment protocol, regimen, process or remedy, treating doesnot require that the desired physiologic response or outcome be achievedin each and every subject or subject population, e.g., patientpopulation. Accordingly, a given subject or subject population, e.g.,patient population, may fail to respond or respond inadequately totreatment.

As used herein, the terms “ameliorate”, “ameliorating” and grammaticalvariations thereof mean to decrease the severity of the symptoms of adisease in a subject.

As used herein, a “subject” is a mammal, preferably, a human. Inaddition to humans, categories of mammals within the scope of thepresent invention include, for example, agricultural animals, veterinaryanimals, laboratory animals, etc. Some examples of agricultural animalsinclude cows, pigs, horses, goats, etc. Some examples of veterinaryanimals include dogs, cats, etc. Some examples of laboratory animalsinclude primates, rats, mice, rabbits, guinea pigs, etc.

Suitable and preferred compounds and pharmaceutical compositions for usein this method are as disclosed above in formulas (I)-(III), includingthe particular SRS compounds.

In one aspect of this embodiment, the disorder is a degenerative diseasethat involves lipid peroxidation. As used herein, “lipid peroxidation”means the oxidative degradation of fats, oils, waxes, sterols,triglycerides, and the like. Lipid peroxidation has been linked withmany degenerative diseases, such as atherosclerosis,ischemia-reperfusion, heart failure, Alzheimer's disease, rheumaticarthritis, cancer, and other immunological disorders. (Ramana et al.,2013).

In another aspect of this embodiment, the disorder is an excitotoxicdisease involving oxidative cell death. As used herein, an “excitotoxicdisorder” means a disease related to the death of central neurons thatare mediated by excitatory amino acids (such as glutamate). Excitotoxicdisorders within the scope of the present invention include diseasesinvolving oxidative cell death. As used herein, “oxidative” cell deathmeans cell death associated with increased levels of intracellularreactive oxygen species (ROS). In the present invention, “reactiveoxygen species” means chemically reactive molecules, such as freeradicals, containing oxygen. Non-limiting examples of ROS include oxygenions and peroxides.

Non-limiting examples of disorders according to the present inventioninclude epilepsy, kidney disease, stroke, myocardial infarction, type Idiabetes, traumatic brain injury (TBI), periventricular leukomalacia(PVL), and neurodegenerative disease. Non-limiting examples ofneurodegenerative diseases according to the present invention includeAlzheimer's, Parkinson's, Amyotrophic lateral sclerosis, Friedreich'sataxia, Multiple sclerosis, Huntington's Disease, Transmissiblespongiform encephalopathy, Charcot-Marie-Tooth disease, Dementia withLewy bodies, Corticobasal degeneration, Progressive supranuclear palsy,and Hereditary spastic paraparesis.

In another aspect of this embodiment, the method further comprisesco-administering, together with one or more compounds or pharmaceuticalcompositions of the present invention, to the subject an effectiveamount of one or more of additional therapeutic agents such as5-hydroxytryptophan, Activase, AFQ056 (Novartis Corp., New York, N.Y.),Aggrastat, Albendazole, alpha-lipoic acid/L-acetyl carnitine, Alteplase,Amantadine (Symmetrel), amlodipine, Ancrod, Apomorphine (Apokyn),Arimoclomol, Arixtra, Armodafinil, Ascorbic acid, Ascriptin, Aspirin,atenolol, Avonex, baclofen (Lioresal), Banzel, Benztropine (Cogentin),Betaseron, BGG492 (Novartis Corp., New York, N.Y.), Botulinum toxin,Bufferin, Carbatrol®, Carbidopa/levodopa immediate-release (Sinemet),Carbidopa/levodopa oral disintegrating (Parcopa),Carbidopa/levodopa/Entacapone (Stalevo), CERE-110: Adeno-AssociatedVirus Delivery of NGF (Ceregene, San Diego, Calif.), cerebrolysin,CinnoVex, citalopram, citicoline, Clobazam, Clonazepam, Clopidogrel,clozapine (Clozaril), Coenzyme Q, Creatine, dabigatran, dalteparin,Dapsone, Davunetide, Deferiprone, Depakene®, Depakote ER®, Depakote®,Desmoteplase, Diastat, Diazepam, Digoxin, Dilantin®, Dimebon,dipyridamole, divalproex (Depakote), Donepezil (Aricept), EGb 761,Eldepryl, ELND002 (Elan Pharmaceuticals, Dublin, Ireland), Enalapril,enoxaparin, Entacapone (Comtan), epoetin alfa, Eptifibatide,Erythropoietin, Escitalopram, Eslicarbazepine acetate, Esmolol,Ethosuximide, Ethyl-EPA (Miraxion™), Exenatide, Extavia, Ezogabine,Felbamate, Felbatol®, Fingolimod (Gilenya), fluoxetine (Prozac),fondaparinux, Fragmin, Frisium, Gabapentin, Gabitril®, Galantamine,Glatiramer (Copaxone), haloperidol (Haldol), Heparin, human chorionicgonadotropin (hCG), Idebenone, Inovelon®, insulin, Interferon beta 1a,Interferon beta 1b, ioflupane 123I (DATSCAN®), IPX066 (ImpaxLaboratories Inc., Hayward, Calif.), JNJ-26489112 (Johnson and Johnson,New Brunswick, N.J.), Keppra®, Klonopin, Lacosamide, L-Alphaglycerylphosphorylcholine, Lamictal®, Lamotrigine, Levetiracetam,liraglutide, Lisinopril, Lithium carbonate, Lopressor, Lorazepam,losartan, Lovenox, Lu AA24493, Luminal, LY450139 (Eli Lilly,Indianapolis, Ind.), Lyrica, Masitinib, Mecobalamin, Memantine,methylprednisolone, metoprolol tartrate, Minitran, Minocycline,mirtazapine, Mitoxantrone (Novantrone), Mysoline®, Natalizumab(Tysabri), Neurontin®, Niacinamide, Nitro-Bid, Nitro-Dur, nitroglycerin,Nitrolingual, Nitromist, Nitrostat, Nitro-Time, Norepinephrine (NOR),Carbamazepine, octreotide, Onfi®, Oxcarbazepine, Oxybutinin chloride,PF-04360365 (Pfizer, New York, N.Y.), Phenobarbital, Phenytek®,Phenytoin, piclozotan, Pioglitazone, Plavix, Potiga, Pramipexole(Mirapex), pramlintide, Prednisone, Primidone, Prinivil, probenecid,Propranolol, PRX-00023 (EPIX Pharmaceuticals Inc.), PXT3003, Quinacrine,Ramelteon, Rasagiline (Azilect), Rebif, ReciGen, remacemide,Resveratrol, Retavase, reteplase, riluzole (Rilutek), Rivastigmine(Exelon), Ropinirole (Requip), Rotigotine (Neupro), Rufinamide, Sabril,safinamide (EMD Serono, Rockland, Mass.), Salagen, Sarafem, Selegiline(1-deprenyl, Eldepryl), SEN0014196 (Siena Biotech, Siena, Italy),sertraline (Zoloft), Simvastatin, Sodium Nitroprussiate (NPS), sodiumphenylbutyrate, Stanback Headache Powder, Tacrine (Cognex), Tamoxifen,tauroursodeoxycholic acid (TUDCA), Tegretol®, Tenecteplase, Tenormin,Tetrabenazine (Xenazine), THR-18 (Thrombotech Ltd.), Tiagabine,Tideglusib, tirofiban, tissue plasminogen activator (tPA), tizanidine(Zanaflex), TNKase, Tolcapone (Tasmar), Tolterodine, Topamax®,Topiramate, Trihexyphenidyl (formerly Artane), Trileptal®, ursodiol,Valproic Acid, valsartan, Varenicline (Pfizer), Vimpat, Vitamin E,Warfarin, Zarontin®, Zestril, Zonegran®, Zonisamide, Zydis selegilineHCL Oral disintegrating (Zelapar), and combinations thereof.

For example, to treat or ameliorate the effects of epilepsy, a subjectmay be administered an effective amount of one or more compounds orpharmaceutical compositions of the present invention and, e.g., one ormore of the following: Albendazole, Banzel, BGG492 (Novartis Corp., NewYork, N.Y.) Carbamazepine, Carbatrol®, Clobazam, Clonazepam, Depakene®,Depakote®, Depakote ER®, Diastat, Diazepam, Dilantin®, Eslicarbazepineacetate, Ethosuximide, Ezogabine, Felbatol®, Felbamate, Frisium,Gabapentin, Gabitril®, Inovelon®, JNJ-26489112 (Johnson and Johnson, NewBrunswick, N.J.) Keppra®, Keppra XR™, Klonopin, Lacosamide, Lamictal®,Lamotrigine, Levetiracetam, Lorazepam, Luminal, Lyrica, Mysoline®,Memantine, Neurontin®, Onfi®, Oxcarbazepine, Phenobarbital, Phenytek®,Phenytoin, Potiga, Primidone, probenecid, PRX-00023 (EPIXPharmaceuticals Inc, Lexington, Mass.), Rufinamide, Sabril, Tegretol®,Tegretol XR®, Tiagabine, Topamax®, Topiramate, Trileptal®, ValproicAcid, Vimpat, Zarontin®, Zonegran®, and Zonisamide.

To treat or ameliorate the effects of stroke, a subject may beadministered an effective amount of one or more compounds orpharmaceutical compositions of the present invention and, e.g., one ormore of the following: Aspirin, dipyridamole, Clopidogrel, tissueplasminogen activator (tPA), Warfarin, dabigatran, Heparin, Lovenox,citicoline, L-Alpha glycerylphosphorylcholine, cerebrolysin,Eptifibatide, Escitalopram, Tenecteplase, Alteplase, Minocycline,Esmolol, Sodium Nitroprussiate (NPS), Norepinephrine (NOR), Dapsone,valsartan, Simvastatin, piclozotan, Desmoteplase, losartan, amlodipine,Ancrod, human chorionic gonadotropin (hCG), epoetin alfa (EPO),Galantamine, and THR-18 (Thrombotech Ltd., Ness Ziona, Israel).

To treat or ameliorate the effects of myocardial infarction, a subjectmay be administered an effective amount of one or more compounds orpharmaceutical compositions of the present invention and, e.g., one ormore of the following: lisinopril, atenolol, Plavix, metoprololtartrate, Lovenox, Lopressor, Zestril, Tenormin, Prinivil, aspirin,Arixtra, clopidogrel, Salagen, nitroglycerin, metoprolol tartrate,heparin, Nitrostat, Nitro-Bid, Stanback Headache Powder, nitroglycerin,Activase, Nitrolingual, nitroglycerin, fondaparinux, Lopressor, heparin,nitroglycerin TL, Nitro-Time, Nitromist, Ascriptin, alteplase, Retavase,TNKase, Bufferin, Nitro-Dur, Minitran, reteplase, tenecteplase,clopidogrel, Fragmin, enoxaparin, dalteparin, tirofiban, and Aggrastat.

To treat or ameliorate the effects of type I diabetes, a subject may beadministered an effective amount of one or more compounds orpharmaceutical compositions of the present invention and, e.g., one ormore of the following: insulin, such as regular insulin (Humulin R,Novolin R, others), insulin isophane (Humulin N, Novolin N), insulinlispro (Humalog), insulin aspart (NovoLog), insulin glargine (Lantus)and insulin detemir (Levemir), octreotide, pramlintide, and liraglutide.

To treat or ameliorate the effects of Alzheimer's disease, a subject maybe administered an effective amount of one or more compounds orpharmaceutical compositions of the present invention and, e.g., one ormore of the following: Donepezil (Aricept), Rivastigmine (Exelon),Galantamine (Razadyne), Tacrine (Cognex), Memantine (Namenda), VitaminE, CERE-110: Adeno-Associated Virus Delivery of NGF (Ceregene), LY450139(Eli Lilly), Exenatide, Varenicline (Pfizer), PF-04360365 (Pfizer),Resveratrol, and Donepezil (Eisai Korea).

To treat or ameliorate the effects of Parkinson's disease, a subject maybe administered an effective amount of one or more compounds orpharmaceutical compositions of the present invention and, e.g., one ormore of the following: Carbidopa/levodopa immediate-release (Sinemet),Carbidopa/levodopa oral disintegrating (Parcopa),Carbidopa/levodopa/Entacapone (Stalevo), Ropinirole (Requip),Pramipexole (Mirapex), Rotigotine (Neupro), Apomorphine (Apokyn),Selegiline (1-deprenyl, Eldepryl), Rasagiline (Azilect), Zydisselegiline HCL Oral disintegrating (Zelapar), Entacapone (Comtan),Tolcapone (Tasmar), Amantadine (Symmetrel), Trihexyphenidyl (formerlyArtane), Benztropine (Cogentin), IPX066 (Impax Laboratories Inc.),Rasagiline (Teva Neuroscience, Inc.), ioflupane 123I (DATSCAN®),safinamide (EMD Serono), and Pioglitazone.

To treat or ameliorate the effects of amyotrophic lateral sclerosis, asubject may be administered an effective amount of one or more compoundsor pharmaceutical compositions of the present invention and, e.g., oneor more of the following: riluzole (Rilutek), Lithium carbonate,Arimoclomol, Creatine, Tamoxifen, Mecobalamin, Memantine (Ebixa), andtauroursodeoxycholic acid (TUDCA).

To treat or ameliorate the effects of Friedreich's ataxia, a subject maybe administered an effective amount of one or more compounds orpharmaceutical compositions of the present invention and, e.g., one ormore of the following: Idebenone, Coenzyme Q, 5-hydroxytryptophan,Propranolol, Enalapril, Lisinopril, Digoxin, Erythropoietin, Lu AA24493,Deferiprone, Varenicline, IVIG, Pioglitazone, and EGb 761.

To treat or ameliorate the effects of multiple sclerosis, a subject maybe administered an effective amount of one or more compounds orpharmaceutical compositions of the present invention and, e.g., one ormore of the following: Avonex, Betaseron, Extavia, Rebif, Glatiramer(Copaxone), Fingolimod (Gilenya), Natalizumab (Tysabri), Mitoxantrone(Novantrone), baclofen (Lioresal), tizanidine (Zanaflex),methylprednisolone, CinnoVex, ReciGen, Masitinib, Prednisone, Interferonbeta 1a, Interferon beta 1b, and ELND002 (Elan Pharmaceuticals).

To treat or ameliorate the effects of Huntington's disease, a subjectmay be administered an effective amount of one or more compounds orpharmaceutical compositions of the present invention and, e.g., one ormore of the following: Tetrabenazine (Xenazine), haloperidol (Haldol),clozapine (Clozaril), clonazepam (Klonopin), diazepam (Valium),escitalopram (Lexapro), fluoxetine (Prozac, Sarafem), sertraline(Zoloft), valproic acid (Depakene), divalproex (Depakote), lamotrigine(Lamictal), Dimebon, AFQ056 (Novartis), Ethyl-EPA (Miraxion™),SEN0014196 (Siena Biotech), sodium phenylbutyrate, citalopram, ursodiol,minocycline, remacemide, and mirtazapine.

To treat or ameliorate the effects of transmissible spongiformencephalopathy, a subject may be administered an effective amount of oneor more compounds or pharmaceutical compositions of the presentinvention and e.g., Quinacrine.

To treat or ameliorate the effects of Charcot-Marie-Tooth disease, asubject may be administered an effective amount of one or more compoundsor pharmaceutical compositions of the present invention and, e.g., oneor more of the following: ascorbic acid and PXT3003.

To treat or ameliorate the effects of dementia with Lewy bodies, asubject may be administered an effective amount of one or more compoundsor pharmaceutical compositions of the present invention and, e.g., oneor more of the following: Aricept, Galantamine, Memantine, Armodafinil,Donepezil, and Ramelteon.

To treat or ameliorate the effects of corticobasal degeneration, asubject may be administered an effective amount of one or more compoundsor pharmaceutical compositions of the present invention and, e.g., oneor more of the following: Davunetide and Coenzyme Q10.

To treat or ameliorate the effects of progressive supranuclear palsy, asubject may be administered an effective amount of one or more compoundsor pharmaceutical compositions of the present invention and, e.g., oneor more of the following: Tideglusib, Rasagiline, alpha-lipoicacid/L-acetyl carnitine, Riluzole, Niacinamide, and Rivastigmine.

To treat or ameliorate the effects of hereditary spastic paraparesis, asubject may be administered an effective amount of one or more compoundsor pharmaceutical compositions of the present invention and, e.g., oneor more of the following: Baclofen, Tizanidine, Oxybutinin chloride,Tolterodine, and Botulinum toxin.

In the present invention, one or more compounds or pharmaceuticalcompositions may be co-administered to a subject in need thereoftogether in the same composition, simultaneously in separatecompositions, or as separate compositions administered at differenttimes, as deemed most appropriate by a physician.

An additional embodiment of the present invention is a method fortreating or ameliorating the effects of a disorder in a subject in needthereof. This method comprises administering to the subject an effectiveamount of a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier or diluent and a compound having the structure offormula (I):

wherein:

-   -   R₁ and R₂, are independently selected from the group consisting        of no atom, H, D, O, halo, C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl, wherein the C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl may be optionally substituted with an        atom or a group selected from the group consisting of halo,        deuterium, C₁₋₄alkyl, CF₃, and combinations thereof; and    -   R₃ is independently selected from the group consisting of H,        C₁₋₁₂aliphatic, C₁₋₆-alkyl-aryl and C₁₋₆-alkyl-heteroaryl;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof,        with the proviso that:    -   when R₃ is ethyl, R₁ and R₂ cannot be both H, O, or

-   -    and    -   when R₃ is ethyl and at least one of R₁ or R₂ is H, R₁ or R₂        cannot be

Suitable and preferred pharmaceutical compositions for use in thismethod are as disclosed above in formulas (I)-(III), includingpharmaceutical compositions containing the particular SRS compounds.Suitable and preferred subjects who may be treated in accordance withthis method are as disclosed above. In this embodiment, the methods maybe used to treat disorders set forth above, including degenerativediseases that involve lipid peroxidation and excitotoxic diseases thatinvolve oxidative cell death.

In another aspect of this embodiment, the method further comprisesco-administering to the subject an effective amount of one or moreadditional therapeutic agents disclosed herein.

Another embodiment of the present invention is a method of modulatingferroptosis in a subject in need thereof. This method comprisesadministering to the subject an effective amount of a ferroptosisinhibitor, which comprises a compound having the structure of formula(I):

wherein:

-   -   R₁ and R₂, are independently selected from the group consisting        of no atom, H, D, O, halo, C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl, wherein the C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl may be optionally substituted with an        atom or a group selected from the group consisting of halo,        deuterium, C₁₋₄alkyl, CF₃, and combinations thereof; and    -   R₃ is independently selected from the group consisting of H,        C₁₋₁₂aliphatic, C₁₋₆-alkyl-aryl and C₁₋₆-alkyl-heteroaryl;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof,        with the proviso that:    -   when R₃ is ethyl, R₁ and R₂ cannot be both H, O, or

-   -    and    -   when R₃ is ethyl and at least one of R₁ or R₂ is H, R₁ or R₂        cannot be

As used herein, “ferroptosis” means regulated cell death that isiron-dependent. Ferroptosis is characterized by the overwhelming,iron-dependent accumulation of lethal lipid reactive oxygen species.(Dixon et al., 2012) Ferroptosis is distinct from apoptosis, necrosis,and autophagy. (Id.) Assays for ferroptosis are as disclosed herein, forinstance, in the Examples section.

Suitable and preferred compounds for use in this method are as disclosedabove in formulas (I)-(III), including the particular SRS compounds.Suitable and preferred subjects who may be treated in accordance withthis method are as disclosed above. In this embodiment, the methods maybe used to treat the disorders set forth above, including degenerativediseases that involve lipid peroxidation and excitotoxic diseases thatinvolve oxidative cell death.

In another aspect of this embodiment, the method further comprisesco-administering to the subject an effective amount of one or moreadditional therapeutic agents disclosed herein.

A further embodiment of the present invention is a method of reducingreactive oxygen species (ROS) in a cell. This method comprisescontacting a cell with a ferroptosis modulator, which comprises acompound having the structure of formula (I):

wherein:

-   -   R₁ and R₂, are independently selected from the group consisting        of no atom, H, D, O, halo, C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl, wherein the C₁₋₆alkyl, C₁₋₆alkyl-aryl,        C₁₋₆alkyl-heteroaryl, C₁₋₆alkenyl, C₁₋₆alkenyl-aryl, and        C₁₋₆alkenyl-heteroaryl may be optionally substituted with an        atom or a group selected from the group consisting of halo,        deuterium, C₁₋₄alkyl, CF₃, and combinations thereof; and    -   R₃ is independently selected from the group consisting of H,        C₁₋₁₂aliphatic, C₁₋₆-alkyl-aryl and C₁₋₆-alkyl-heteroaryl;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof,        with the proviso that:    -   when R₃ is ethyl, R₁ and R₂ cannot be both H, O, or

-   -    and    -   when R₃ is ethyl and at least one of R₁ or R₂ is H, R₁ or R₂        cannot be

As used herein, the terms “modulate”, “modulating”, “modulator” andgrammatical variations thereof mean to change, such as decreasing orreducing the occurrence of ferroptosis. In this embodiment, “contacting”means bringing the compound and optionally one or more additionaltherapeutic agents into close proximity to the cells in need of suchmodulation. This may be accomplished using conventional techniques ofdrug delivery to the subject or in the in vitro situation by, e.g.,providing the compound and optionally other therapeutic agents to aculture media in which the cells are located.

Suitable and preferred compounds for use in this method are as disclosedabove in formulas (I)-(III), including the particular SRS compounds. Inthis embodiment, reducing ROS may be accomplished in cells obtained froma subject having a disorder as disclosed herein. Suitable and preferredsubjects of this embodiment are as disclosed above.

In one aspect of this embodiment, the cell is a mammalian cell.Preferably, the mammalian cell is obtained from a mammal selected fromthe group consisting of humans, primates, farm animals, and domesticanimals. More preferably, the mammalian cell is a human cancer cell.

In another aspect of this embodiment, the method further comprisescontacting the cell with at least one additional therapeutic agent asdisclosed herein.

An additional embodiment of the present invention is a method fortreating or ameliorating the effects of a neurodegenerative disease in asubject in need thereof. This method comprises administering to thesubject an effective amount of a compound selected from the groupconsisting of

and combinations thereof, or an N-oxide, crystalline form, hydrate, orpharmaceutically acceptable salt thereof.

Suitable and preferred subjects are as disclosed herein. In thisembodiment, the methods may be used to treat the neurodegenerativedisorders set forth above.

In one aspect of this embodiment, the method further comprisesco-administering to the subject an effective amount of one or moretherapeutic agents disclosed herein.

Another embodiment of the present invention is a compound according toformula (IV):

wherein:

-   -   R₁ is selected from the group consisting of aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle, wherein the aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle may be optionally        substituted with an atom or a group selected from the group        consisting of halo, deuterium, C₁₋₆alkyl, CF₃, and combinations        thereof;    -   R₂ is a triazole, an oxazole, an oxadiazole, or a ketone; and    -   R₃ is a C₃₋₁₂carbocycle, optionally substituted with C₁₋₁₀ alkyl        or halo;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof.

In one aspect of this embodiment, the compound has the structure offormula (V):

wherein:

-   -   R₁ is selected from the group consisting of aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle, wherein the aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle may be optionally        substituted with an atom or a group selected from the group        consisting of halo, deuterium, C₁₋₆alkyl, CF₃, and combinations        thereof; and    -   R₂ is selected from the group consisting of

-   -   wherein R₄ is selected from the group consisting of H,        C₁₋₁₂alkyl, C₃₋₁₂carbocycle, and aryl,        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof.

In another aspect of this embodiment, R1 is selected from the groupconsisting of:

wherein R₅ is selected from the group consisting of CF₃, CH₃, CH₂CH₃,CH(CH₃)₂, C(CH₃)₃;or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptablesalt thereof.

In a further aspect of this embodiment, the compound has the structureof:

or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptablesalt thereof.

A further embodiment of the present invention is a pharmaceuticalcomposition. This pharmaceutical composition comprises apharmaceutically acceptable carrier or diluent and a compound accordingto formula (IV):

wherein:

-   -   R₁ is selected from the group consisting of aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle, wherein the aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle may be optionally        substituted with an atom or a group selected from the group        consisting of halo, deuterium, C₁₋₆alkyl, CF₃, and combinations        thereof;    -   R₂ is a triazole, an oxazole, an oxadiazole, or a ketone; and    -   R₃ is a C₃₋₁₂carbocycle, optionally substituted with C₁₋₁₀ alkyl        or halo;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof.

Suitable and preferred compounds, including preferred R1 groups,compounds of formula (V), and SRS13-10F2, are as set forth above.

An additional embodiment of the present invention is a kit. This kitcomprises a compound or a pharmaceutical composition according to anycompound of the present invention together with instructions for the useof the compound.

Suitable and preferred compounds, including preferred R₁ groups,compounds of formula (V), and SRS13-10F2, are as set forth above.

Another embodiment of the present invention is a method for treating orameliorating the effects of a disorder in a subject in need thereof.This method comprises administering to the subject an effective amountof a compound having the structure of formula (IV):

wherein:

-   -   R₁ is selected from the group consisting of aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle, wherein the aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle may be optionally        substituted with an atom or a group selected from the group        consisting of halo, deuterium, C₁₋₆alkyl, CF₃, and combinations        thereof;    -   R₂ is a triazole, an oxazole, an oxadiazole, or a ketone; and    -   R₃ is a C₃₋₁₂carbocycle, optionally substituted with C₁₋₁₀ alkyl        or halo;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof.

Suitable and preferred compounds, including preferred R₁ groups,compounds of formula (V), and SRS13-10F2, are as set forth above.Suitable and preferred subjects are also as set forth above.

In one aspect of this embodiment, the disorder is a degenerative diseasethat involves lipid peroxidation. In another aspect of this embodiment,the disorder is an excitotoxic disease involving oxidative cell death.The disorder may also be selected from the group consisting of epilepsy,kidney disease, stroke, myocardial infarction, type I diabetes, TBI,PVL, and neurodegenerative disease. Suitable and preferredneurodegenerative diseases are as set forth above.

In a further aspect of this embodiment, the method further comprisesco-administering to the subject an effective amount of one or moreadditional therapeutic agents as disclosed herein.

A further embodiment of the present invention is a method for treatingor ameliorating the effects of a disorder in a subject in need thereof.This method comprises administering to the subject an effective amountof a pharmaceutical composition comprising a pharmaceutically acceptablecarrier or diluent and a compound having the structure of formula (IV):

wherein:

-   -   R₁ is selected from the group consisting of aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle, wherein the aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle may be optionally        substituted with an atom or a group selected from the group        consisting of halo, deuterium, C₁₋₆alkyl, CF₃, and combinations        thereof;    -   R₂ is a triazole, an oxazole, an oxadiazole, or a ketone; and    -   R₃ is a C₃₋₁₂carbocycle, optionally substituted with C₁₋₁₀ alkyl        or halo;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof.

Suitable and preferred compounds, including preferred R₁ groups,compounds of formula (V), and SRS13-10F2, are as set forth above.Suitable and preferred subjects are also as set forth above.

In one aspect of this embodiment, the disorder is a degenerative diseasethat involves lipid peroxidation. In another aspect of this embodiment,the disorder is an excitotoxic disease involving oxidative cell death.The disorder may also be selected from the group consisting of epilepsy,kidney disease, stroke, myocardial infarction, type I diabetes, TBI,PVL, and neurodegenerative disease. Suitable and preferredneurodegenerative diseases are as set forth above.

Another embodiment of the present invention is a method of modulatingferroptosis in a subject in need thereof. This method comprisesadministering to the subject an effective amount of a ferroptosisinhibitor, which comprises a compound having the structure of formula(IV):

wherein:

-   -   R₁ is selected from the group consisting of aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle, wherein the aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle may be optionally        substituted with an atom or a group selected from the group        consisting of halo, deuterium, C₁₋₆alkyl, CF₃, and combinations        thereof;    -   R₂ is a triazole, an oxazole, an oxadiazole, or a ketone; and    -   R₃ is a C₃₋₁₂carbocycle, optionally substituted with C₁₋₁₀ alkyl        or halo;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof.

Suitable and preferred compounds, including preferred R₁ groups,compounds of formula (V), and SRS13-10F2, are as set forth above.Suitable and preferred subjects are also as set forth above.

An additional embodiment of the present invention is a method ofreducing reactive oxygen species (ROS) in a cell. This method comprisescontacting a cell with a ferroptosis modulator, which comprises acompound having the structure of formula (IV):

wherein:

-   -   R₁ is selected from the group consisting of aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle, wherein the aryl,        C₁₋₆alkyl-aryl, and C₃₋₁₀carbocycle may be optionally        substituted with an atom or a group selected from the group        consisting of halo, deuterium, C₁₋₆alkyl, CF₃, and combinations        thereof;    -   R₂ is a triazole, an oxazole, an oxadiazole, or a ketone; and    -   R₃ is a C₃₋₁₂carbocycle, optionally substituted with C₁₋₁₀ alkyl        or halo;        or an N-oxide, crystalline form, hydrate, or pharmaceutically        acceptable salt thereof.

Suitable and preferred compounds, including preferred R₁ groups,compounds of formula (V), and SRS13-10F2, are as set forth above.

In one aspect of this embodiment, the cell may be accomplished in cellsobtained from a subject having a disease that involves lipidperoxidation. Suitable and preferred subjects are as set forth above.Preferably, the disorder is an excitotoxic disease involving oxidativecell death. The disorder may also be selected from the group consistingof epilepsy, kidney disease, stroke, myocardial infarction, type Idiabetes, TBI, PVL, and neurodegenerative disease. Suitable andpreferred neurodegenerative diseases are as set forth above.

In one aspect of this embodiment, the cell is a mammalian cell.Preferably, the mammalian cell is obtained from a mammal selected fromthe group consisting of humans, primates, farm animals, and domesticanimals. More preferably, the mammalian cell is a human cancer cell.

As used herein, a “pharmaceutically acceptable salt” means a salt of thecompounds of the present invention which are pharmaceuticallyacceptable, as defined herein, and which possess the desiredpharmacological activity. Such salts include acid addition salts formedwith inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like; or withorganic acids such as acetic acid, propionic acid, hexanoic acid,heptanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid,lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,o-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,p-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,p-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid,4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid and the like. Pharmaceuticallyacceptable salts also include base addition salts which may be formedwhen acidic protons present are capable of reacting with inorganic ororganic bases. Acceptable inorganic bases include sodium hydroxide,sodium carbonate, potassium hydroxide, aluminum hydroxide and calciumhydroxide. Acceptable organic bases include ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine and thelike.

In the present invention, an “effective amount” or “therapeuticallyeffective amount” of a compound or pharmaceutical composition, is anamount of such a compound or composition that is sufficient to effectbeneficial or desired results as described herein when administered to asubject. Effective dosage forms, modes of administration, and dosageamounts may be determined empirically, and making such determinations iswithin the skill of the art. It is understood by those skilled in theart that the dosage amount will vary with the route of administration,the rate of excretion, the duration of the treatment, the identity ofany other drugs being administered, the age, size, and species of thesubject, and like factors well known in the arts of, e.g., medicine andveterinary medicine. In general, a suitable dose of a compound orpharmaceutical composition according to the invention will be thatamount of the compound or composition, which is the lowest doseeffective to produce the desired effect with no or minimal side effects.The effective dose of a compound or pharmaceutical composition accordingto the present invention may be administered as two, three, four, five,six or more sub-doses, administered separately at appropriate intervalsthroughout the day.

A suitable, non-limiting example of a dosage of a compound orpharmaceutical composition according to the present invention or acomposition comprising such a compound, is from about 1 ng/kg to about1000 mg/kg, such as from about 1 mg/kg to about 100 mg/kg, includingfrom about 5 mg/kg to about 50 mg/kg. Other representative dosages of acompound or a pharmaceutical composition of the present inventioninclude about 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg,30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg,250 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800mg/kg, 900 mg/kg, or 1000 mg/kg.

A compound or pharmaceutical composition of the present invention may beadministered in any desired and effective manner: for oral ingestion, oras an ointment or drop for local administration to the eyes, or forparenteral or other administration in any appropriate manner such asintraperitoneal, subcutaneous, topical, intradermal, inhalation,intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous,intraarterial, intrathecal, or intralymphatic. Further, a compound orpharmaceutical composition of the present invention may be administeredin conjunction with other treatments. A compound or pharmaceuticalcomposition of the present invention may be encapsulated or otherwiseprotected against gastric or other secretions, if desired.

The pharmaceutical compositions of the invention are pharmaceuticallyacceptable and comprise one or more active ingredients in admixture withone or more pharmaceutically-acceptable carriers or diluents and,optionally, one or more other compounds, drugs, ingredients and/ormaterials. Regardless of the route of administration selected, thecompounds/pharmaceutical compositions of the present invention areformulated into pharmaceutically-acceptable dosage forms by conventionalmethods known to those of skill in the art. See, e.g., Remington, TheScience and Practice of Pharmacy (21^(st) Edition, Lippincott Williamsand Wilkins, Philadelphia, Pa.). More generally, “pharmaceuticallyacceptable” means that which is useful in preparing a composition thatis generally safe, non-toxic, and neither biologically nor otherwiseundesirable and includes that which is acceptable for veterinary use aswell as human pharmaceutical use.

Pharmaceutically acceptable carriers and diluents are well known in theart (see, e.g., Remington, The Science and Practice of Pharmacy (21^(st)Edition, Lippincott Williams and Wilkins, Philadelphia, Pa.) and TheNational Formulary (American Pharmaceutical Association, Washington,D.C.)) and include sugars (e.g., lactose, sucrose, mannitol, andsorbitol), starches, cellulose preparations, calcium phosphates (e.g.,dicalcium phosphate, tricalcium phosphate and calcium hydrogenphosphate), sodium citrate, water, aqueous solutions (e.g., saline,sodium chloride injection, Ringer's injection, dextrose injection,dextrose and sodium chloride injection, lactated Ringer's injection),alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol),polyols (e.g., glycerol, propylene glycol, and polyethylene glycol),organic esters (e.g., ethyl oleate and tryglycerides), biodegradablepolymers (e.g., polylactide-polyglycolide, poly(orthoesters), andpoly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils(e.g., corn, germ, olive, castor, sesame, cottonseed, and groundnut),cocoa butter, waxes (e.g., suppository waxes), paraffins, silicones,talc, silicylate, etc. Each pharmaceutically acceptable carrier ordiluent used in a composition of the invention must be “acceptable” inthe sense of being compatible with the other ingredients of theformulation and not injurious to the subject. Carriers or diluentssuitable for a selected dosage form and intended route of administrationare well known in the art, and acceptable carriers or diluents for achosen dosage form and method of administration can be determined usingordinary skill in the art.

The pharmaceutical compositions of the invention may, optionally,contain additional ingredients and/or materials commonly used in suchcompositions. These ingredients and materials are well known in the artand include (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and silicic acid; (2) binders, such ascarboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, suchas glycerol; (4) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,sodium starch glycolate, cross-linked sodium carboxymethyl cellulose andsodium carbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,and sodium lauryl sulfate; (10) suspending agents, such as ethoxylatedisostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agarand tragacanth; (11) buffering agents; (12) excipients, such as lactose,milk sugars, polyethylene glycols, animal and vegetable fats, oils,waxes, paraffins, cocoa butter, starches, tragacanth, cellulosederivatives, polyethylene glycol, silicones, bentonites, silicic acid,talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, andpolyamide powder; (13) inert diluents, such as water or other solvents;(14) preservatives; (15) surface-active agents; (16) dispersing agents;(17) control-release or absorption-delaying agents, such ashydroxypropylmethyl cellulose, other polymer matrices, biodegradablepolymers, liposomes, microspheres, aluminum monosterate, gelatin, andwaxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21)emulsifying and suspending agents; (22), solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan; (23)propellants, such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane; (24) antioxidants; (25) agentswhich render the formulation isotonic with the blood of the intendedrecipient, such as sugars and sodium chloride; (26) thickening agents;(27) coating materials, such as lecithin; and (28) sweetening,flavoring, coloring, perfuming and preservative agents. Each suchingredient or material must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the subject. Ingredients and materials suitable for aselected dosage form and intended route of administration are well knownin the art, and acceptable ingredients and materials for a chosen dosageform and method of administration may be determined using ordinary skillin the art.

Compounds or pharmaceutical compositions suitable for oraladministration may be in the form of capsules, cachets, pills, tablets,powders, granules, a solution or a suspension in an aqueous ornon-aqueous liquid, an oil-in-water or water-in-oil liquid emulsion, anelixir or syrup, a pastille, a bolus, an electuary or a paste. Theseformulations may be prepared by methods known in the art, e.g., by meansof conventional pan-coating, mixing, granulation or lyophilizationprocesses.

Solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like) may be prepared, e.g., bymixing the active ingredient(s) with one or morepharmaceutically-acceptable carriers or diluents and, optionally, one ormore fillers, extenders, binders, humectants, disintegrating agents,solution retarding agents, absorption accelerators, wetting agents,absorbents, lubricants, and/or coloring agents. Solid compositions of asimilar type may be employed as fillers in soft and hard-filled gelatincapsules using a suitable excipient. A tablet may be made by compressionor molding, optionally with one or more accessory ingredients.Compressed tablets may be prepared using a suitable binder, lubricant,inert diluent, preservative, disintegrant, surface-active or dispersingagent. Molded tablets may be made by molding in a suitable machine. Thetablets, and other solid dosage forms, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient therein.They may be sterilized by, for example, filtration through abacteria-retaining filter. These compositions may also optionallycontain opacifying agents and may be of a composition such that theyrelease the active ingredient only, or preferentially, in a certainportion of the gastrointestinal tract, optionally, in a delayed manner.The active ingredient can also be in microencapsulated form.

Liquid dosage forms for oral administration includepharmaceutically-acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. The liquid dosage forms may containsuitable inert diluents commonly used in the art. Besides inertdiluents, the oral compositions may also include adjuvants, such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents. Suspensions maycontain suspending agents.

Compositions for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing one or more activeingredient(s) with one or more suitable nonirritating carriers which aresolid at room temperature, but liquid at body temperature and,therefore, will melt in the rectum or vaginal cavity and release theactive compound. Compositions which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such pharmaceutically-acceptablecarriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, drops and inhalants. The active agent(s)/compound(s) may bemixed under sterile conditions with a suitablepharmaceutically-acceptable carrier or diluent. The ointments, pastes,creams and gels may contain excipients. Powders and sprays may containexcipients and propellants.

Compositions suitable for parenteral administrations comprise one ormore agent(s)/compound(s) in combination with one or morepharmaceutically-acceptable sterile isotonic aqueous or non-aqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain suitable antioxidants,buffers, solutes which render the formulation isotonic with the blood ofthe intended recipient, or suspending or thickening agents. Properfluidity can be maintained, for example, by the use of coatingmaterials, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants. These compositions mayalso contain suitable adjuvants, such as wetting agents, emulsifyingagents and dispersing agents. It may also be desirable to includeisotonic agents. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption.

In some cases, in order to prolong the effect of a drug (e.g.,pharmaceutical formulation), it is desirable to slow its absorption fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material havingpoor water solubility.

The rate of absorption of the active agent/drug then depends upon itsrate of dissolution which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of aparenterally-administered agent/drug may be accomplished by dissolvingor suspending the active agent/drug in an oil vehicle. Injectable depotforms may be made by forming microencapsule matrices of the activeingredient in biodegradable polymers. Depending on the ratio of theactive ingredient to polymer, and the nature of the particular polymeremployed, the rate of active ingredient release can be controlled. Depotinjectable formulations are also prepared by entrapping the drug inliposomes or microemulsions which are compatible with body tissue. Theinjectable materials can be sterilized for example, by filtrationthrough a bacterial-retaining filter.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier or diluent, for example water for injection, immediately priorto use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets of the typedescribed above.

In the foregoing embodiments, the following definitions apply.

The term “aliphatic”, as used herein, refers to a group composed ofcarbon and hydrogen that do not contain aromatic rings. Accordingly,aliphatic groups include alkyl, alkenyl, alkynyl, and carbocyclylgroups. Additionally, unless otherwise indicated, the term “aliphatic”is intended to include both “unsubstituted aliphatics” and “substitutedaliphatics”, the latter of which refers to aliphatic moieties havingsubstituents replacing a hydrogen on one or more carbons of thealiphatic group. Such substituents can include, for example, a halogen,a deuterium, a hydroxyl, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, aphosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine,an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, asulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, aheterocyclyl, an aralkyl, an aromatic, or heteroaromatic moiety.

The term “alkyl” refers to the radical of saturated aliphatic groupsthat does not have a ring structure, including straight-chain alkylgroups, and branched-chain alkyl groups. In certain embodiments, astraight chain or branched chain alkyl has 6 or fewer carbon atoms inits backbone (e.g., C₁-C₆ for straight chains, C₃-C₆ for branchedchains). Such substituents include all those contemplated for aliphaticgroups, as discussed below, except where stability is prohibitive.

The term “alkenyl”, as used herein, refers to an aliphatic groupcontaining at least one double bond and unless otherwise indicated, isintended to include both “unsubstituted alkenyls” and “substitutedalkenyls”, the latter of which refers to alkenyl moieties havingsubstituents replacing a hydrogen on one or more carbons of the alkenylgroup. Such substituents include all those contemplated for aliphaticgroups, as discussed below, except where stability is prohibitive. Forexample, substitution of alkenyl groups by one or more alkyl,carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.

Moreover, unless otherwise indicated, the term “alkyl” as usedthroughout the specification, examples, and claims is intended toinclude both “unsubstituted alkyls” and “substituted alkyls”, the latterof which refers to alkyl moieties having substituents replacing ahydrogen on one or more carbons of the hydrocarbon backbone. Indeed,unless otherwise indicated, all groups recited herein are intended toinclude both substituted and unsubstituted options.

The term “C_(x-y)” when used in conjunction with a chemical moiety, suchas, alkyl and cycloalkyl, is meant to include groups that contain from xto y carbons in the chain. For example, the term “C_(x-y)alkyl” refersto substituted or unsubstituted saturated hydrocarbon groups, includingstraight-chain alkyl and branched-chain alkyl groups that contain from xto y carbons in the chain, including haloalkyl groups such astrifluoromethyl and 2,2,2-tirfluoroethyl, etc.

The term “aryl” as used herein includes substituted or unsubstitutedsingle-ring aromatic groups in which each atom of the ring is carbon.Preferably the ring is a 3- to 8-membered ring, more preferably a6-membered ring. The term “aryl” also includes polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings is aromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groupsinclude benzene, naphthalene, phenanthrene, phenol, aniline, and thelike.

The term “alkyl-aryl” refers to an alkyl group substituted with at leastone aryl group.

The term “alkyl-heteroaryl” refers to an alkyl group substituted with atleast one heteroaryl group.

The term “alkenyl-aryl” refers to an alkenyl group substituted with atleast one aryl group.

The term “alkenyl-heteroaryl” refers to an alkenyl group substitutedwith at least one heteroaryl group.

The terms “carbocycle”, “carbocyclyl”, and “carbocyclic”, as usedherein, refer to a non-aromatic saturated or unsaturated ring in whicheach atom of the ring is carbon. Preferably a carbocycle ring containsfrom 3 to 10 atoms, more preferably from 3 to 8 atoms, including 5 to 7atoms, such as for example, 6 atoms.

The terms “halo” and “halogen” are used interchangeably herein and meanhalogen and include chloro, fluoro, bromo, and iodo.

The term “heteroaryl” includes substituted or unsubstituted aromaticsingle ring structures, preferably 3- to 8-membered rings, morepreferably 5- to 7-membered rings, even more preferably 5- to 6-memberedrings, whose ring structures include at least one heteroatom, preferablyone to four heteroatoms, more preferably one or two heteroatoms. Theterm “heteroaryl” also includes polycyclic ring systems having two ormore cyclic rings in which two or more carbons are common to twoadjoining rings wherein at least one of the rings is heteroaromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroarylgroups include, for example, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, andpyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, andsulfur; more preferably, nitrogen and oxygen.

The term “ketone” means an organic compound with the structure RC(═O)R′,wherein neither R and R′ can be hydrogen atoms.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with the permitted valence ofthe substituted atom and the substituent, and that the substitutionresults in a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this invention, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include any substituents described herein,for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, aphosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine,an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, asulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, aheterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate.

As set forth previously, unless specifically stated as “unsubstituted,”references to chemical moieties herein are understood to includesubstituted variants. For example, reference to an “aryl” group ormoiety implicitly includes both substituted and unsubstituted variants.

As used herein, the term “oxadiazole” means any compound or chemicalgroup containing the following structure:

As used herein, the term “oxazole” means any compound or chemical groupcontaining the following structure:

As used herein, the term “triazole” means any compound or chemical groupcontaining the following structure:

It is understood that the disclosure of a compound herein encompassesall stereoisomers of that compound. As used herein, the term“stereoisomer” refers to a compound made up of the same atoms bonded bythe same bonds but having different three-dimensional structures whichare not interchangeable. The three-dimensional structures are calledconfigurations. Stereoisomers include enantiomers and diastereomers.

The terms “racemate” or “racemic mixture” refer to a mixture of equalparts of enantiomers. The term “chiral center” refers to a carbon atomto which four different groups are attached. The term “enantiomericenrichment” as used herein refers to the increase in the amount of oneenantiomer as compared to the other.

It is appreciated that to the extent compounds of the present inventionhave a chiral center, they may exist in and be isolated in opticallyactive and racemic forms. Some compounds may exhibit polymorphism. It isto be understood that the present invention encompasses any racemic,optically-active, diastereomeric, polymorphic, or stereoisomeric form,or mixtures thereof, of a compound of the invention, which possess theuseful properties described herein, it being well known in the art howto prepare optically active forms (for example, by resolution of theracemic form by recrystallization techniques, by synthesis fromoptically-active starting materials, by chiral synthesis, or bychromatographic separation using a chiral stationary phase).

Examples of methods to obtain optically active materials are known inthe art, and include at least the following:

-   -   i) physical separation of crystals—a technique whereby        macroscopic crystals of the individual enantiomers are manually        separated. This technique can be used if crystals of the        separate enantiomers exist, i.e., the material is a        conglomerate, and the crystals are visually distinct;    -   ii) simultaneous crystallization—a technique whereby the        individual enantiomers are separately crystallized from a        solution of the racemate, possible only if the latter is a        conglomerate in the solid state;    -   iii) enzymatic resolutions—a technique whereby partial or        complete separation of a racemate by virtue of differing rates        of reaction for the enantiomers with an enzyme;    -   iv) enzymatic asymmetric synthesis—a synthetic technique whereby        at least one step of the synthesis uses an enzymatic reaction to        obtain an enantiomerically pure or enriched synthetic precursor        of the desired enantiomer;    -   v) chemical asymmetric synthesis—a synthetic technique whereby        the desired enantiomer is synthesized from an achiral precursor        under conditions that produce asymmetry (i.e., chirality) in the        product, which may be achieved using chiral catalysts as        disclosed in more detail herein or chiral auxiliaries;    -   vi) diastereomer separations—a technique whereby a racemic        compound is reacted with an enantiomerically pure reagent (the        chiral auxiliary) that converts the individual enantiomers to        diastereomers. The resulting diastereomers are then separated by        chromatography or crystallization by virtue of their now more        distinct structural differences and the chiral auxiliary later        removed to obtain the desired enantiomer;    -   vii) first- and second-order asymmetric transformations—a        technique whereby diastereomers from the racemate equilibrate to        yield a preponderance in solution of the diastereomer from the        desired enantiomer or where preferential crystallization of the        diastereomer from the desired enantiomer perturbs the        equilibrium such that eventually in principle all the material        is converted to the crystalline diastereomer from the desired        enantiomer. The desired enantiomer is then released from the        diastereomer;    -   viii) kinetic resolutions—this technique refers to the        achievement of partial or complete resolution of a racemate (or        of a further resolution of a partially resolved compound) by        virtue of unequal reaction rates of the enantiomers with a        chiral, non-racemic reagent or catalyst under kinetic        conditions;    -   ix) enantiospecific synthesis from non-racemic precursors—a        synthetic technique whereby the desired enantiomer is obtained        from non-chiral starting materials and where the stereochemical        integrity is not or is only minimally compromised over the        course of the synthesis;    -   x) chiral liquid chromatography—a technique whereby the        enantiomers of a racemate are separated in a liquid mobile phase        by virtue of their differing interactions with a stationary        phase. The stationary phase can be made of chiral material or        the mobile phase can contain an additional chiral material to        provoke the differing interactions;    -   xi) chiral gas chromatography—a technique whereby the racemate        is volatilized and enantiomers are separated by virtue of their        differing interactions in the gaseous mobile phase with a column        containing a fixed non-racemic chiral adsorbent phase;    -   xii) extraction with chiral solvents—a technique whereby the        enantiomers are separated by virtue of preferential dissolution        of one enantiomer into a particular chiral solvent;    -   xiii) transport across chiral membranes—a technique whereby a        racemate is placed in contact with a thin membrane barrier. The        barrier typically separates two miscible fluids, one containing        the racemate, and a driving force such as concentration or        pressure differential causes preferential transport across the        membrane barrier. Separation occurs as a result of the        non-racemic chiral nature of the membrane which allows only one        enantiomer of the racemate to pass through.

The stereoisomers may also be separated by usual techniques known tothose skilled in the art including fractional crystallization of thebases or their salts or chromatographic techniques such as LC or flashchromatography. The (+) enantiomer can be separated from the (−)enantiomer using techniques and procedures well known in the art, suchas that described by J. Jacques, et al., Enantiomers, Racemates, andResolutions”, John Wiley and Sons, Inc., 1981. For example, chiralchromatography with a suitable organic solvent, such asethanol/acetonitrile and Chiralpak AD packing, 20 micron can also beutilized to effect separation of the enantiomers.

The following examples are provided to further illustrate the methods ofthe present invention. These examples are illustrative only and are notintended to limit the scope of the invention in any way.

EXAMPLES Example 1 Experimental Procedures

Flow Cytometry Experiments

200,000 HT-1080 cells were seeded into 6-well dishes (Corning). The nextday, cells were treated with +/−rotenone +/−ferrostatin-1 for 3 hours(for MitoSOX experiments) or +/−STS +/−ferrostatin-1 for 4 hours (for10-N-nonylacridine orange [NAO] experiments), harvested bytrypsinization and analyzed as follows. For the analysis ofmitochondrial ROS, cells were incubated with 5 μM MitoSOX (MolecularProbes/Invitrogen) in HBSS for 10 minutes in the dark at 37° C. For theanalysis of mitochondrial cardiolipin peroxidation, cells were incubatedwith NAO dissolved to a final concentration of 25 ng/mL in HBSS for 10minutes in the dark at 37° C. Cells were then washed once in HBSS andmeasurements were subsequently acquired using an flow cytometer(BD/Accuri C6) equipped with a 488 nm laser for excitation offluorophores. MitoSOX data were collected in the FL2 channel and NAOdata was collected in the FL1 channel. Data are from three independentbiological replicates of each experiment. Data were analyzed by one-wayANOVA with Bonferroni's post-hoc test.

Analysis of Acridine Orange (AO) Relocalization

200,000 HT-1080 cells were seeded into 6-well dishes (Corning). The nextday, cells were incubated with 25 ng/mL of AO for 15 minutes, thenmedium was removed and cells were incubated in growth medium for 15minutes. The medium was then removed again and replaced with mediumcontaining DMSO or erastin +/−ciclipirox olamine (CPX, an iron chelator)or ferrostatin-1 (Fer-1). Relocalization of AO from lysosomes (discretered puncta) to the cytosol (diffuse green color) was determined using anEVOS fl digital inverted fluorescent microscope (Advanced MicroscopyGroup) equipped with GFP (ex/em 470/525) and RFP (ex/em 531/593) lightcubes.

Analysis of Reactive Oxygen Species Production

The day before the experiment, 200,000 cells/well were seeded in 6-welldishes (Corning Inc., Corning, N.Y.). The day of the experiment, cellswere treated with test compounds for the indicated times, then harvestedby trypsinization, resuspended in 500 μL Hanks Balanced Salt Solution(HBSS, Gibco, Invitrogen Corp., Carlsbad, Calif.) containing eitherH₂DCFDA (25 μM), C11-BODIPY(581/591) (2 μM) or MitoSOX (5 μM) (all fromMolecular Probes, Invitrogen) and incubated for 10 minutes at 37° C. ina tissue culture incubator. Cells were then resuspended in 500 μL offresh HBSS, strained through a 40 μM cell strainer (BD Falcon, BDBiosciences, San Jose, Calif.), and analyzed using a flow cytometer(FACSCalibur or Accuri C6, BD Biosciences), which was equipped with488-nm laser for excitation. Data was collected from the FL1 (H₂DCFDA,C11-BODIPY) or FL2 channel (MitoSOX). A minimum of 10,000 cells wereanalyzed per condition.

Cancer Cell Viability Measurements

Cell viability was typically assessed in 384-well format by Alamar Blue(Invitrogen) fluorescence (ex/em 530/590) measured on a Victor3platereader (Perkin Elmer, Waltham, Mass.). In some experiments, TrypanBlue dye exclusion counting was performed using an automated cellcounter (ViCell, Beckman-Coulter Inc., Brea, Calif.). Cell viability intest conditions is reported as a percentage relative to the negativecontrol treatment.

shRNA Screening

An arrayed collection of 6,528 shRNA hairpins derived from The RNAiConsortium (TRC) collection targeting 1,087 genes, kindly provided byVamsi Mootha and Joshua Baughman (MIT), was screened in 384-well plateformat (Corning) using both Calu-1 and HT-1080 cells. ShRNAs targetingGFP and RFP, randomly distributed through each plate, served as negativecontrols. 400 cells per well were infected in duplicate for 48 hourswith 2 μL shRNA-containing viral supernatant, selected for 24 hours inpuromycin (1.5 μg/mL), then treated with DMSO, erastin (7.3 μM) orstaurosporine (STS) (1 μM) for 24 hours. Cell viability was determinedusing Alamar Blue. For each hairpin within each treatment condition, acell death rescue score was computed as the ratio of the averageviability of the two replicates to the average viability of thewithin-plate negative controls. These scores were used to compare theeffects between compounds. To identify genes required for ferroptosis,individual hairpins were scored as hits if they displayed an averagedeath suppression ≥3 median average deviations from the medianwithin-plate or screen-wide negative control values. 51 candidate geneswere identified with the same two (or more) unique hairpins per genecalled as hits in both the Calu-1 and HT-1080 screens. For eachcandidate gene, confirmation studies using RT-qPCR analysis of mRNAsilencing was performed in HT-1080 cells using freshly prepared virus asdescribed in more detail below.

[¹⁴C]Cystine Uptake Assay

200,000 HT-1080 cells/well were seeded overnight in 6-well dishes(Corning). The next day, cells were washed twice in pre-warmed Na⁺-freeuptake buffer (137 mM choline chloride, 3 mM KCl, 1 mM CaCl₂, 1 mMMgCl₂, 5 mM D-glucose, 0.7 mM K₂HPO₄, 10 mM HEPES, pH 7.4), thenincubated for 10 minutes at 37° C. in 1 mL of uptake buffer, to depletecellular amino acids. At this point, in each well the buffer wasreplaced with 600 μL uptake buffer containing compound and 0.12 μCi(80-110 mCi/mmol) of L-[3,3′-¹⁴C]-cystine (Perkin Elmer) and incubatedfor 3 minutes at 37° C. Cells were then washed three times with ice-colduptake buffer and lysed in 500 μL 0.1 M NaOH. To this lysate was added15 mL of scintillation fluid and radioactive counts per minute wereobtained using a scintillation counter. All measurements were performedin triplicate for each condition.

Statistical Analyses

All statistical analyses were performed using Prism 5.0c (GraphPadSoftware Inc., La Jolla, Calif.).

Chemicals

Erastin was synthesized as described (Yagoda et al., 2007). RSL3 wasobtained from Leadgen Laboratories (Orange, Conn.). Rapamycin wasobtained from Cell Signaling Technologies (Danvers, Mass.), z-VAD-fmkwas from BioMol (Enzo Life Sciences, Inc., Farmingdale, N.Y.), ALLN andE64D were from CalBiochem (Merck KGaA, Darmstadt, Germany), bafilomycinA1 and U0126 were from LC Laboratories (Woburn, Mass.), BAPTA-AM andFura-2 were from Invitrogen. GKT137831 was the generous gift ofGenKyoTex S.A. (Geneva, Switzerland). Unless otherwise indicated, allother compounds were from Sigma (St. Louis, Mo.).

Cell Lines and Media

The following engineered human foreskin fibroblasts were obtained fromRobert Weinberg (Whitehead Institute): BJeH, BJeHLT, BJeLR, DRD. BJeHcells express human telomerase (hTERT), BJeHLT express hTERT plus thelarge and small T oncoproteins (LT, ST), BJeLR express hTERT, LT, ST andan oncogenic HRAS allele (HRAS^(V12)), DRD cells express an alternativesuite of oncoproteins: hTERT, ST, dominant-negative p53, cyclin D1, anda mutant form of CDK4, along with HRAS^(V12). MEFs (Bax^(−/−)Bak^(−/−)and wild-type) were obtained from Craig Thompson (Sloan Kettering),143B.TK-mtDNA-depleted rho zero (ρ⁰) and matching parental ρ⁺ cells wereobtained from Eric Schon (Columbia University), TC32 and SK-ES-1 wereobtained from Stephen Lessnick (Huntsman Cancer Institute, Salt LakeCity). HT-1080, Calu-1, U2OS and 293-T cells were obtained from AmericanType Culture Collection.

BJ series cell lines were grown in DMEM High-Glucose media (Gibco,Invitrogen Corp., Carlsbad, Calif.) plus 20% M199 (Sigma) and 15%heat-inactivated fetal bovine serum (FBS). HT-1080 cells were grown inDMEM High-Glucose media (Gibco) supplemented with 10% FBS and 1%non-essential amino acids (Gibco). Calu-1 and U2OS cells were grown inMcCoy's 5A media (Gibco) supplemented with 10% fetal bovine serum.SK-ES-1 cells were grown in McCoy's 5A supplemented with 1.5 mML-glutamine+15% FBS. 293-T and TC32 cells were grown in DMEMHigh-Glucose supplemented with 10% FBS. When used for transfections togenerate virus, 293-T cells were seeded in the above media lackingantibiotics. 293-T viral collection media contained 30% HyClone FBS.MEFs were grown in DMEM supplemented with 10% fetal calf serum. 143Bcells were grown in DMEM High-Glucose supplemented with 10% FBS. 143B ρ⁰cells were grown in the above media supplemented with 100 μg/mL uridine.The rho zero status of the 143B ρ⁰ cell lines was confirmed usingRT-qPCR by showing little or no mRNA expression for 7 mtDNA-encodedtranscripts in the ρ⁰ cell lines. All cell lines were grown inhumidified tissue culture incubators (Thermo Scientific) at 37° C. with5% CO₂. Except where indicated, all medias were supplemented withpenicillin and streptomycin (Gibco).

Light Microscopy

Phase contrast images were acquired using an AMG EVOS FL (AdvancedMicroscopy Group) microscope equipped with a 10× phase-contrastobjective. Three independent fields were acquired for each experimentalcondition. Representative samples from one field of view are shown.

Transmission Electron Microscopy

BJeLR cells were plated at 100,000 cells/dish in 35 mm tissue culturedishes. After 12 hours, cells were treated with vehicle (DMSO; 10hours), erastin (37 μM; 10 hours), staurosporine (750 nM; 8 hours),hydrogen peroxide (16 mM; 1 hour) or rapamycin (100 nM; 24 hours). Cellswere fixed with 2.5% glutaraldehyde in 0.1 M Sorenson's buffer (0.1 MH₂PO₄, 0.1 M HPO₄ (pH 7.2)) for at least 1 hour, and then treated with1% 050₄ in 0.1 M Sorenson's buffer for 1 hour. Enblock staining used 1%tannic acid. After dehydration through an ethanol series, cells wereembedded in Lx-112 (Ladd Research Industries, Williston, Vt.) andEmbed-812 (Electron Microscopy Sciences, Hatfield, Pa.). Thin sectionswere cut on an MT-7000 ultramicrotome, stained with 1% uranyl acetateand 0.4% lead citrate, and examined under a Jeol JEM-1200 EXII electronmicroscope. Pictures were taken on an ORCA-HR digital camera (HamamatsuCorp., Bridgewater, N.J.) at 5,000-50,000-fold magnification, andmeasurements were made using the AMT Image Capture Engine.

Measurement of ATP Levels

ATP levels were evaluated using the ApoSENSOR ATP Assay Kit (BiovisionInc., Milpitas, Calif.) according to the manufacturer's instructions.2000 HT-1080 or BJeLR cells were seeded in 96-well white bottom plates(Falcon). Cells were treated 12 hours later with compound as above forthe TEM. Prior to luminescence measurement, medium was removed and cellswere lysed and incubated with ATP Monitoring Enzyme. Luminescence wasmeasured using a Victor3 platereader equipped with an infrared emissionfilter every 30 seconds for 10 minutes. Typically the first reading wasused for data analysis. Parallel cell culture treatments were performedin 96-well clear bottom plates and cell viability was determined usingAlamar Blue. These values were used to normalize ATP levels to cellviability.

Modulatory Profiling of Small Molecule Inhibitors

The following small molecule inhibitors were tested in a ten-point,four-fold dilution series for their ability to prevent erastin-induceddeath in HT-1080, Calu-1 and BJ-eLR cells (high dose of dilution seriesin brackets):carbobenzyloxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone(z-VAD-fmk, 184 μM), necrostatin-1 (Nec-1, 19.3 μM), cyclosporine A(CspA, 33.2 μM), N-Acetyl-Leu-Leu-Nle-CHO (ALLN, 40 μM),(2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester(E64d, 400 μM), bafilomycin A1 (Baf A1, 4 μM), 3-methyladenine (3-MA,6.25 mM), chloroquine (Chq, 250 μM), deferoxamine (DFO, 400),6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, 320 μM),1,4-diamino-2,3-dicyano-1,4-bis (2-aminophenylthio)butadiene (U0126,52.4 μM) and cycloheximide (5.6 μM). The day before the experiment,cells were seeded in black, 384-well clear bottom plates (Corning) at adensity of 1,000 cells/well using a Beckman Biomek FX Workstation. Allcompounds (except 3-methyladenine and chloroquine, which were diluted inwater) were diluted in DMSO at 250 times the final highest testconcentration, and aliquoted across a 384-well plate (Greiner, Monroe,N.C.). A ten-point, 4-fold dilution series of each inhibitor in DMSO wasmade using a multichannel pipette. Replicates of this mother plate werestored at −20° C. On the day of the experiment, a fresh inhibitor platewas thawed and diluted 25-fold into DMEM in a 384 deep well ‘daughter’plate (Greiner), using a Biomek FX. Medium was removed from cells andreplaced with 36 μL medium containing DMSO (0.1%) or erastin (10 μMfinal concentration). Using the Biomek FX, 4 μL were then transferredfrom each inhibitor daughter plate into the DMSO-containing orerastin-containing assay plates. For 3-methyladenine and chloroquine,inhibitors were prepared fresh in media at 10-fold the finalconcentration in the presence of DMSO or 10 μM erastin. These solutionswere manually pipetted onto cells in 384-well format. Assay plates wereincubated at 37° C. for 24 hours. Viability was assessed using AlamarBlue.

Computation of Modulatory Effects

The modulatory effect (M_(e)) for drug-drug interactions was computed asfollows. First, plate-based background correction was performed usingAlamar Blue values from empty (cell-free) wells included on each plate.Next, within each experiment, 100% viability is determined as theaverage Alamar Blue score for cells treated with modulator=DMSO andlethal=DMSO, and all Alamar Blue values were scaled from 0 to 1 with theDMSO×DMSO condition=1. The individual effects of each modulator andlethal (e.g. in the presence of DMSO) at all tested doses were thenascertained. Using these data, the “expected” effect on viability(B_(Exp)) for each modulator (M)×lethal (L) combination were modeledusing the Bliss formula for drug interactions (M+L-M*L). Next, B_(Exp)was compared to the actual observed viability. For each informativedrug-drug interaction (i.e. modulator=“DFO”, modulator concentration=100μM, lethal=Erastin) the modulatory effect (M_(e)) was equivalent to themaximum observed deviation, positive or negative, from B_(Exp). Bydefault, M_(e) in DMSO-treated cells is equal to 0. This formula wasempirically determined to provide a useful measure of modulation that isrobust to differences in the shapes of individual dose-response curvesfor different inhibitors and lethal molecules. M_(e) values for eachinhibitor-lethal drug combination were hierarchically clustered andplotted as a heatmap using the heatmaps.2 function of the gplots libraryin R.

Modulatory Profile of Lethal Small Molecules

The following lethal molecules were compared in a ten-point, two-folddilution series modulatory profiling assay (high dose of dilution seriesin brackets): erastin (20 μM), RSL3 (5 μM), hydrogen peroxide (H₂0₂, 5mM), artesunate (200 μM), phenylarsine oxide (PAO, 1 μM), taxol (0.5μM), suberoylanilide hydroxamic acid (SAHA, 50 μM), trichostatin A (2.5μM), doxorubicin (2 μM), fenretinide (50 μM), staurosporine (STS, 0.5μM), brefeldin A (10 μM), β-lapachone (25 μM), bortezomib (5 μM),carbonyl cyanide m-chlorophenyl hydrazine (CCCP, 50 μM),2-methoxyestradiol (2-ME, 50 μM), rotenone (2 μM), helenaline (50 μM),sulfasalazine (SAS, 1 mM) and phenethyl isothiocyanate (PEITC, 50 μM).

The comparative analysis of the lethal effects of various molecules wasconducted as above for the inhibitors experiment with the followingmodifications. All lethal molecules were tested in a ten point, two-folddilution series. Inhibitors were made in media at 1× finalconcentration. The growth media was removed from the plates, and 36 μLmedia+inhibitor (or DMSO) was added back to the plate+4 μL from the 10×lethal stock plate. Cell viability and modulatory effects (M_(e)) werecomputed as above, to obtain the maximum deviation from B_(EXp) producedby each modulator across the different lethal small moleculeconcentrations.

Arrayed shRNA Screen: Data Analysis and Hit Selection.

ShRNA screening and follow-up studies identified 51 initial candidatesuppressor genes, each represented by 2 hairpins common to both celllines. These candidates were re-tested. For 50 of these genes, freshvirus (see below) was prepared for the top two scoring shRNA hairpins.The resistance in HT-1080 cells infected with these hairpins in responseto up to 10 μM erastin was re-tested. In parallel, target knockdown wasvalidated for each shRNA hairpin by RT-qPCR. From the initial set ofsuppressor genes, high confidence genes were selected using thefollowing criteria: (1) the degree of death suppression was consistentacross two independent replicates in the re-testing phase; (2) the levelof death suppression was at least 50% of that observed in thesh263-VDAC3 positive control at the highest dose of erastin (10 μM); (3)at least one of two shRNA hairpins must reduce the level of mRNA<50% ofcontrol, and (4) an inverse correlation must exist between erastinresistance and mRNA levels (providing a strong measure of confidence inthe on-target nature of the shRNAs used). Finally, whether each hairpinhad been reported as independently validated was determined on The RNAiConsortium/Sigma website. By intersecting the results of theseconfirmatory analyses, a final set of six high confidence genes wasdetermined.

Arrayed shRNA Screen: shRNA Confirmation

For all candidate suppressors, individual “hit” shRNA hairpin sequenceswere non-overlapping (e.g. targeting unique sequences within the mRNA)and were confirmed using the siRNA-Check tool available from In SilicoSolutions (Fairfax, Va.). Individual shRNAs are identified by the 3 or 4number Clone ID suffixes assigned to each mRNA target sequences by TRC.

New virus were produced to validate erastin resistance and performdownstream analyses of target knockdown and functional effects in a6-well format as follows. On Day 1, 170,000 293-T cells were seeded inantibiotic-free media into each well of a 6-well dish. On Day 2, thesecells were transfected (Fugene, Roche) with 450 ng of shRNA plasmid DNA,400 ng of pDelta8.9 helper plasmid and 45 ng of pVSVg helper plasmid. OnDay 3, the media was switched to viral collection media. Virus washarvested the following morning and evening of Day 4 and then a finaltime the next morning of Day 5. The collected media was pooled, spun at2,000 rpm for 5 minutes and the virus-containing supernatant aliquotedand stored at −80° C. This protocol was used for the production of virusin all other small-scale shRNA experiments as well (i.e. FIGS. 7C and7D).

Six-well dish infections were performed as follows. On Day 1, 30,000HT-1080 cells were seeded per well. On day 2, cells were infected with150 μL of viral supernatant and spin infected as for 384-well plates.

Genetic Screening Follow-up Experiments: Cell Line and Lethal CompoundSpecificity Analysis

These experiments were performed for all cell lines. Lethal compoundexperiments were performed in parallel by first re-arraying by handvirus prepared as described above for all suppressor hairpins in asingle 384 deep well viral “mother plate” (Corning), including 3independent copies of the negative control and positive controls(sh-Control and sh263-VDAC3). Each hairpin was arrayed in a block of 6wells (2 across×3 down). For each experiment, cells were seeded at astandard density of 400 cells/well on Day 1. On Day 2, the media wasremoved either by flicking, (for HT-1080 cells in the lethal compoundanalysis) or using a BioMek FX (for the cell line analysis), and thenreplaced with 38 μl media containing 1× polybrene (8 μg/ml) using theBioMek. 2 μl viral soup was then transferred from each well of the viralmother plate to each well of the assay plate and a spin infection wasperformed as described above. On Day 3 (for the analysis of cell lines)or Day 4 (for the analysis of lethal compounds), the media+virus wasremoved using the BioMek and replaced with media+1.5 μg/ml puromycin.Next, for both experiments, on Day 5, the media was again removed usingthe BioMek and replaced with media+lethal compound. On Day 6, AlamarBlue was added as described above and the signal was measured 6 hourslater. This experiment was repeated twice with similar results andrepresentative data from one experiment is shown. Data for the besthairpin as defined by mRNA silencing levels in HT-1080 cells isdisclosed herein. Similar results were obtained with the second besthairpins.

Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR)

RNA was extracted using the Qiashredder and Qiagen RNeasy Mini kits(Qiagen) according to the manufacturer's protocol. 2 μg total RNA foreach sample was used as input for each reverse transcription reaction,performed using the TaqMan RT kit (Applied Biosystems, Life TechnologiesCorp., Carlsbad, Calif.). Primer pairs were designed for targettranscripts using Primer Express 2.0 (Applied Biosystems). QuantitativePCR reactions were performed using the Power SYBR Green PCR Master Mix(Applied Biosystems). Triplicate samples per condition were analyzed onan Applied Biosystems 7300 qPCR instrument using absolute quantificationsettings. Differences in mRNA levels compared to HPRT1 or ACTB internalreference control were computed between control and experimentalconditions using the ΔΔCt method.

LOC Library Construction

The LOC (Lead Optimized Compound) library is composed of 9,517 compoundsselected from a starting pool of 3,372,615 compounds available through avariety of commercial libraries (Asinex, Moscow, Russia; Life Chemicals,Burlington, ON, Canada; Enamine Ltd., Kiev, Ukraine; TimTec, Newark,Del.; InterBioScreen Ltd., Moscow, Russia; Chembridge Corp., San Diego,Calif.). The starting pool was generated in silico by downloadingstructure files for available compounds from all vendors. From thispool, the application of Lipinski's rules (Lipinski et al., 2001) andother relevant physicochemical descriptors consistent with drug-likecandidates (molecular weight>235, number of rotatable bonds<5,topological polar surface area<70 Å², aqueous solubility>0.5 mM) reducedthe total number of compounds to 58,786. The total number of compoundswas further reduced to 45,395 by filtering out compounds containingnitro and nitroso groups, reactive moieties, ketones and aldehydes,imines, scaffolds unsuitable for further modification, organometalliccompounds and thiols. From this set, the final collection was derived byeliminating multiple copies of highly similar compounds (Tanimotocoefficient). All computational analyses were performed using MOE2008.10(Chemical Computing Group, Montreal, Canada) on a MacPro with 2×2.93 GHzQuad-Core Intel Xeon CPUs. At this point, 5 mg of each compound wasobtained from their respective suppliers and dissolved in DMSO at astandard concentration of 4 mg/mL. Aliquots of each compound were thenarrayed into individual wells of several 384 shallow well “mother”plates (Grenier) using a BioMek liquid handling robot and frozen at −80°C. until use.

LOC Library Screening

The LOC library was screened over the course of several days. 384-wellglass bottom assay plates (Corning) were seeded with 1,000 HT-1080cells/well the day before the experiment. The day of the experiment, LOCmother plates were thawed at room temperature for 1 hour and spun at1000 rpm for 1 minute prior to use. Using a BioMek liquid handing robot,2 μL of compound solution from each LOC library mother plate wastransferred to a 384 deep well “daughter” plate (Grenier) containing 148μL of cell culture media. The cell culture media in each assay plate wasthen removed by flicking and, using a BioMek FX, replaced with 36 μL ofgrowth media containing erastin (5 μM final). To this was added 4 μL ofthe drug solution from the daughter plate, for a final screeningconcentration of 5.3 μg/mL for each LOC library compound. 4 wellscontaining DMSO alone (no erastin), 4 wells containing 100 μM DFO alone(no erastin), 4 wells containing erastin plus DMSO, and 4 wellscontaining erastin plus 100 μM DFO (positive control) were included ascontrols on each plate. Each drug daughter plate was aliquotedseparately to duplicate assay plates. Plates were then spun briefly(1000 rpm, 5 seconds) and returned to the 37° C. tissue cultureincubator. 24 hours later, cell viability was assessed by Alamar Blue asdescribed above.

LOC Library Screen Hit Identification and Confirmation

Candidate hits from the LOC library screen were identified. First,values for each duplicate screening plate were averaged. Next, a growthrescue score consisting of the ratio of the viability of each LOCcompound+erastin versus the DMSO+erastin treatment within each plate wascomputed. These plate-based growth rescue scores were rank ordered fromhighest to lowest. The top 336 ranked compounds, derived from the sameLOC library plates used for screening, were then re-tested in HT-1080cells in a 10-point, 2-fold dilution series against erastin (5 μM) asdescribed above for the modulatory profiling experiments. For the top 50most potent compound inhibitors of erastin-induced death identified inthis experiment, fresh compound in powder form was re-ordered from therespective vendors, the powder was re-dissolved in DMSO as above, andthe 10-point, 2-fold dilution series experiment in HT-1080 cellsrepeated. Fer-1 proved to be the most potent of the re-tested compoundsin this experiment and was selected for more detailed study.

Western Blotting

Cells were trypsinized, pelleted, and washed once in PBS. Cells werelysed for 20 minutes in buffer containing: 50 mM HEPES pH 7.4, 40 mMNaCl, 2 mM EDTA, 0.5% Triton X-100, 1.5 mM sodium orthovanadate, 50 mMNaF, 10 mM sodium pyrophosphate, 10 mM sodium β-glycerophosphate, andprotease inhibitor tablet (Roche, Nutley, N.J.). Unlysed cells anddebris were pelleted for 10 minutes at 10,000 rpm in a benchtopmicrocentrifuge at 4° C., and the supernatant was removed and mixed with5×SDS loading buffer. Samples were separated by SDS-polyacrylamide gelelectrophoresis. Western transfer was performed using the iBlot system(Invitrogen). Membrane was blocked for 1 hour in Licor Odyssey BlockingBuffer (LI-COR) and incubated in primary antibody overnight at 4° C.Following three 5-minute washes in Tris-buffered saline (pH 7.4) with 1%Tween-20 (TBS-T), membrane was incubated with secondary antibodies for45 minutes in the dark. The membrane was washed again in TBST for three5-minute washes with protection from light and scanned using the OdysseyImaging System (LI-COR). Antibodies used were as follows: rabbitanti-phospho p42/44 MAPK (Cell Signaling Technology, #9101) and rabbitanti-p42/44 MAPK (Cell Signaling Technology, #9102). The secondaryantibody was IR dye 800CW goat anti-rabbit IgG (LI-COR).

2,2-diphenyl-1-picrylhydrazyl (DPPH) Assay

The stable radical DPPH (Blois, 1958) was dissolved in methanol to afinal working concentration of 0.05 mM. This was prepared as follows.First, a 100× stock concentration (5 mM) was prepared by dissolving 3.9mg DPPH in 2 mL methanol. Then, for 25 mL of 0.05 mM final workingsolution, 250 μL of the 5 mM solution was added to 24.75 mL of methanol.1 mL of DPPH solution was added to a small volume (<5 μL) each testcompound dissolved in DMSO. The final concentration of each testcompound was 0.05 mM. Samples were inverted several times and allowed toincubate at room temperature for 30 minutes. Samples were then aliquotedto white 96-well solid-bottom dishes (Corning) and absorbance at 517 nmwas recorded using a TECAN M200 plate reader. All values were normalizedto background (methanol only). The experiment was repeated three timesand the data was averaged.

Analysis of Cell Death in Rat Brain Slices: Organotypic HippocampalSlice Cultures (OHSCs)

OHSCs were cultured as previously described (Morrison et al., 2002) withapproval from Columbia University's Institutional Animal Care and UseCommittee (IACUC). Briefly, Sprague Dawley rat pups (P8-P10) wererapidly decapitated, and the hippocampus placed in ice-cold Gey'sbalanced salt solution (Sigma). 400 μm thick sections were cut using aMcllwain tissue chopper and immediately plated on Millicell cell cultureinserts (Millipore, Billerica, Mass.) in Neurobasal (Invitrogen) mediasupplemented with B27 (1×, Invitrogen), GlutaMAX (1 mM, Invitrogen), andD-glucose (4.5 mg/mL, Sigma) at 37° C. and 5% CO₂. After 2 days in vitro(DIV), the media was changed to medium containing serum comprised of 50%DMEM (Sigma), 25% heat-inactivated horse serum (Sigma), 25% Hank'sbalanced salt solution (Sigma), GlutaMAX (1 mM, Invitrogen), andD-glucose (4.5 mg/mL, Sigma). Medium was changed every 2-3 days.

Analysis of Cell Death in Rat Brain Slices: Excitotoxic Injury

After 10-14 DIV, OBSCs were exposed to an excitotoxic injury consistingof a 3 hour exposure to 5 mM L-glutamate in SFM (Morrison et al., 2002).Only healthy OHSCs defined as those with less than 5% cell death in allregions of the hippocampus (DG, CA3, CA1) pre-injury were used forexperiments. After the 3 hour exposure, the cultures were placed infresh serum free media (SFM) containing 75% DMEM, 25% Hank's balancedsalt solution, GlutaMAX (1 mM), D-glucose (4.5 mg/mL) until cell deathwas quantified at 24 hours. If drugs were added, they were added at thesame time as glutamate.

Analysis of Cell Death in Rat Brain Slices: Cell Death Assessment

Quantification of cell death has been described previously (Cater etal., 2007; Morrison et al., 2002). Brightfield images of the hippocampalcultures were taken before injury for identification of regions ofinterest (ROI) including the dentate gyrus (DG), CA3 and CA1. Propidiumiodide (PI, Invitrogen) was used as a fluorescent signal for cell death,and images were taken before the induction of injury and 24 hoursfollowing injury. For PI imaging, the cultures were transferred to SFMsupplemented with 5 μg/mL PI. After a 30 minute incubation, brightfieldand PI images were acquired. All images were captured on an OlympusIX-80 fluorescent microscope fitted with a 175 W Xenon Arc lamp (PerkinElmer), CoolSNAP ES camera (Photometrics, Tucson, Ariz.), and standardrhodamine optics (excitation 556-580 nm; emission 590-630 nm; PIexposure 2 seconds, brightfield exposure 3 milliseconds). Metamorphimage analysis software was used to determine the ROI in the brightfieldimage, and this ROI was transferred to the PI image taken before and 24hours after injury. Percentage cell death was expressed as the number ofpixels in the ROI above a threshold in the PI fluorescent image dividedby the total number of pixels in the ROI.

Brain Slice Assay for HD

250 μm corticostriatal brain slices were prepared from postnatal day 10CD Sprague-Dawley rat pups (Charles River) as previously described(Hoffstrom et al., 2010). Brain slice explants were placed in interfaceculture in 6-well plates using culture medium containing 15%heat-inactivated horse serum, 10 mM KCl, 10 mM HEPES, 100 U/mlpenicillin/streptomycin, 1 mM MEM sodium pyruvate, and 1 mM L-glutaminein Neurobasal A (Invitrogen) and maintained in humidified incubatorsunder 5% CO₂ at 32 deg. C. A custom-modified biolistic device (HeliosGene Gun; Bio-Rad) was used to transfect the brain slices with a humanhtt exon-1 expression construct containing a 73 CAG repeat (“HttN90Q73”)in the gWiz backbone (Genlantis) together with a YFP expressionconstruct to visualize transfected neurons. Control brain slices weretransfected with gWiz blank vector and YFP at the equivalent DNAamounts. After 4 days of incubation, MSNs were identified by theirlocation within the striatum and by their characteristic dendriticmorphology and scored as healthy if expressing bright and continuous YFPlabeling throughout, normal-sized cell bodies, and >2 primarydendrites >2 cell bodies long, as previously described²¹. Data wereexpressed as mean numbers of healthy MSNs per striatal region in eachbrain slice, with statistical significance tested by ANOVA followed byDunnett's post hoc comparison test at the 0.05 confidence level. Fer-1was added to the culture medium at the time of brain slice preparation;positive control brain slices were treated with a combination of theadenosine receptor 2A modulator KW-6002 (50 μM) and the JNK inhibitorSP600125 (30 μM). Final DMSO concentration of 0.1% for all conditions.

Brain Slice Assay for HD Fer-1 Analogs Protected DevelopingOligodendrocytes from Cystine Deprivation Induced Cell Death

Primary pre-oligodendrocytes cultures were prepared from the forebrainsof P2 Sprague Dawley (Charles River Laboratory) rat pups using adifferential detachment method. Forebrains free of meninges weredissociated with Hanks' Balanced Salt Solution containing 0.01% trypsinand 10 μg/ml DNase, and triturated with DMEM containing 10%heat-inactivated fetal bovine serum and 100 U/ml penicillin and 100μg/ml streptomycin. Dissociated cells were plated ontopoly-D-lysine-coated 75 cm² flasks and fed cells every other day for10-17 days. On day 10 or 17, following 1 hour pre-shake at 200 rpm 37°C. to remove microglia, the flasks were shaken overnight to separatepre-oligodendrocytes from astrocyte layer. The cell suspension waspassed through a 20 μm filter and plated onto uncoated (bacteriological)petri dishes for 1 hour in incubator to remove residualmicroglia/astrocytes. Cell suspension was plated ontopoly-D,L-ornithine-coated plates with DMEM, 1×ITS (Life Technologies), 2mM L-glutamine, 1 mM sodium pyruvate, 0.5% FBS and 0.05% gentamicin(Sigma), 10 ng/ml PDGF and 10 ng/ml FGF (Peprotech), with full mediumchange the next day and half medium change every other day. At day 8,cells were washed twice with cystine deprivation medium, treated withFer-1 and analogs (stock 1 mM in DMSO) in cystine deprivation mediumplus PDGF and FGF (treatment medium) for 24 hrs. Cells were treated withtreatment medium plus 100 μM cystine as positive control; and cells weretreated with treatment medium as negative control. Cells in each well,received same amount of DMSO as a vehicle. After 24 hours, cells wereassayed with Alamar Blue (Treck Diagnostics) by full medium change with1×AlamarBlue in Earle's Balance Salt Solution for 2 hours at 37° C. and5% CO₂. Fluorescence was assayed in each well using FluoroCount PlateReader (Packard), with Packard Plate Reader Version 3.0, and 530 nmexcitation, and 590 nm emission filters.

Studies of Isolated Mouse Proximal Tubules

Tubule preparation: 8-12 week old C57/BL6 female mice were euthanizedwith isoflurane. Kidneys were removed and immediately injectedintraparenchymally with a cold 95% O₂/5% CO₂-gassed solution consistingof 115 mM NaCl, 2.1 mM KCl, 25 mM NaHCO₃, 1.2 mM KH₂PO₄, 2.5 mM CaCl₂,1.2 mM MgCl₂, 1.2 mM MgSO₄, 25 mM mannitol, 2.5 mg/ml fatty acid freebovine serum albumin, 5 mM glucose, 4 mM sodium lactate, I mM alanine,and 1 mM sodium butyrate (Solution A) with the addition of 1 mg/mlcollagenase (Type I, Worthington Biochemical Corp., Freehold). Thecortices were then dissected and minced on an ice cold tile, thenresuspended in additional Solution A for 8-10 min. of digestion at 37°C. followed by enrichment of proximal tubules using centrifugation onself-forming Percoll gradients as previously described for rabbittubules.

Experimental procedures for isolated tubules: Tubules were suspended at1.5-2.0 mg tubule protein/mi in a 95% air/5% CO₂-gassed mediumcontaining (in mM) 110 NaCl, 2.6 KCl, 25 NaHCO₃, 2.4 KH₂PO₄, 1.25 CaCl₂,1.2 MgCl₂, 1.2 MgSO₄, 5 glucose, 4 sodium lactate, 0.3 alanine, 5 sodiumbutyrate, 2 glycine, and 1.0 mg/ml bovine gelatin (75 bloom) (SolutionB). After precincubation for 15 min. at 37° C., then were thenresuspended in fresh Solution B containing 2 mM heptanoic acid insteadof sodium butyrate along with experimental compounds.

Oncogenic-RAS-selective Lethal Assay

Analysis of oncogenic-RAS-selective lethality in BJeH, BJeHLT, BJeLR andDRD cells was performed as described previously (Yang and Stockwell,2008).

siRNA Gene Silencing

All siRNAs were obtained from Qiagen. 50,000 HT-1080 cells were seededin antibiotic-free HT-1080 media into each well of a 6-well tissueculture dish (Corning) the day before the start of the experiment. Thenext day, cells were transfected with 2 nM of siRNAs (finalconcentration/well) using Lipofectamine RNAiMAX (Invitrogen) accordingto the manufacturer's protocols. The media was replaced with freshantibiotic-containing HT-1080 media the following day. Parallel cultureswere assayed for gene expression after 48 hours using RT-qPCR and forcell viability in response to drug treatment at 72 hourspost-transfection.

Plasmids and Transfection

pMaxGFP plasmid was from Amaxa (Lonza Group Ltd., Basel, Switzerland).pCMV6-SLC7A11-DDK was from Origene Technologies (Rockville, Md.). 50,000HT-1080 cells were seeded in 6-well dishes (Corning) the day before theexperiment in regular HT-1080 media. The next day, cells weretransfected with 0.5 μg plasmid DNA/well using Lipofectamine LTX(Invitrogen) according to the manufacturer's protocol. 72 hourspost-transfection, cell viability was assessed by microscopy in responseto erastin and sulfasalazine.

Identification of SLC7A5 as an Erastin Target in BJeH and BJeLR Cells

Affinity chromatography and mass spectrometry were used to identifyproteins that bound an active (lethal) erastin analog (erastin-A6) andan inactive control (erastin-B2) in lysates from oncogenic HRAS-mutantBJeLR cells and HRAS-wild-type BJeH cells (2 independent experimentalreplicates for each cell line) (Yagoda et al., 2007). Previously, theanalyses on targets bound by active erastin-A6 were done in BJeLR versusBJeH cells, on the assumption that such targets were most likely tomediate oncogenic-RAS-selective lethality (Yagoda et al., 2007).However, given that erastin was lethal to various cells, including somethat lack mutant HRAS, the data were re-analyzed in order to look fortargets bound by active erastin-A6 (but not inactive erastin-B2) in bothcells types, as follows. First the two experimental replicates weremerged on the basis of protein reference IDs to identify high confidencetargets for each erastin analog in both cell lines. Any proteins alsobound by inactive erastin-B2 (i.e. non-specific interactions) were theneliminated from the erastin-A6 target lists. Next, the lists of proteinsuniquely bound by active erastin-A6 in BJeH cells (gi|4506675 [RPN1],gi|4505773 [PHB], gi|1174469 [STT3A], gi|14017819 [LRRIQ1], gi|12643412[SLC7A5]) and BJeLR cells (gi|1172554 [VDAC2], gi|19923753 [SLC16A1],gi|11281610 [TECR], gi|23308572 [MBOAT7], gi|4759086 [SEC22B],gi|7448310 [TSPO], gi|4507943 [XPO1], gi|21361181 [ATP1A1], gi|29029559[CSEL1], gi|23308577 [PHGDH], gi|1362789 [PRKDC], gi|12643412 [SLC7A5])were compared. This new analysis identified SLC7A5 as the lone proteinbound by active erastin-A6 in both cell types. Of note, VDAC2 wasidentified in both experimental replicates for erastin-A6-treated BJeLRcells, but annotated with two different identifiers (gi|4507881 andgi|1172554). Also of note, SLC3A2 was identified in one replicate oferastin-A6-treated BJeLR cells.

Analysis of Metabolic Profiling Data for Erastin-treated Jurkat Cells

Ramanathan and Schreiber isolated a total of 123 metabolites from JurkatT cells treated with erastin (1 μM, 25 minutes) or vehicle control(Ramanathan and Schreiber, 2009). These authors previously reported on asubset (11/123) of the metabolites that are specifically related tomitochondrial metabolism and glycolytic pathway function (FIG. 6,(Ramanathan and Schreiber, 2009)). The complete (123 metabolite)normalized dataset was obtained, and the data were ranked by theobserved significance (P values) of the change in abundance betweenerastin-treated and control samples. The substrate specificity of systemL has previously been established (Kanai and Endou, 2003).

Software

Flow cytometry data was analyzed using FloJo (9.3.2, Tree Star, Inc.,Ashland, Oreg.). Chemical structures were drawn using ChemDraw Ultra(Cambridgesoft, Cambridge, Mass.). Computational determination of log P(S log P function) was performed using MOE 2010.10 (Chemical ComputingGroup, Montreal, Calif.). Viability data was analyzed using Excel(Microsoft Corp., Seattle, Oreg.). Summary data and heatmaps weregenerated using R. Dose response curves were computed by 4-parameterlogistic regression in Prism 5.0c (GraphPad Software). Images weremanipulated using Photoshop CS4 and Illustrator CS4 (Adobe, San Jose,Calif.).

Example 2 Synthesis of Ferrostatin-1 Analogs

Chemicals:

Solvents, inorganic salts, and organic reagents were purchased fromcommercial sources such as Sigma and Fisher and used without furtherpurification unless otherwise noted. Erastin was dissolved in DMSO to afinal concentration of 73.1 mM and stored in aliquots at −20° C.

Chromatography:

Merck pre-coated 0.25 mm silica plates containing a 254 nm fluorescenceindicator were used for analytical thin-layer chromatography. Flashchromatography was performed on 230-400 mesh silica (SiliaFlash® P60)from Silicycle.

Spectroscopy:

¹H, ¹³C and ¹⁹F NMR spectra were obtained on a Bruker DPX 400 MHzspectrometer. HRMS spectra were taken on double focusing sector typemass spectrometer HX-110A. Maker JEOL Ltd. Tokyo Japan (resolution of10,000 and 10 KV accel. Volt. Ionization method; FAB (Fast AtomBombardment) used Xe 3Kv energy. Used Matrix, NBA (m-Nitro benzylalcohol)).

General Procedure A (ArS_(N)2 Reaction) (Beaulieu et al. 2003)

To ethyl 4-chloro-3-nitrobenzoate (1 equiv., 300 mg, 1.3 mmol) in dryDMSO (5 mL) was added K₂CO₃ (2 equiv., 360 mg, 1.739 mmol) and1-adamantylamine (1.2 equiv., 238 mg, 1.572 mmol). The mixture wasstirred for 17 hours at 60° C. The solution was poured into water andthe organic layer was extracted three times with ethyl acetate. Afterdrying with anhydrous magnesium sulfate the solvents were removed undervacuum. The residue was purified by flash-column chromatography onsilica gel to provide the desired ethyl4-(adamanthyl-amino)-3-nitrobenzoate derivatives (Scheme 3; Scheme 4,SRS-16-79, R_(a)=tert-butyl, MW=372).

General Procedure B (Hydrogenolysis)

The ethyl 4-(adamanthyl-amino)-3-nitrobenzoates (130 mg, 0.445 mmol)were dissolved in MeOH (10 mL) and hydrogenated (H₂ gas) over 10%Pd(OH)₂ on charcoal (90 mg) for 17 hours at room temperature. Thesolution was filtered through a pad of celite and volatiles were removedunder vacuum. The residue was purified by flash-column chromatography onsilica gel to provide the desired Ferrostatin-1 analogs.

General Procedure C (Reductive Amination Reaction) (Abdel-Magid et al.,1996)

Method I: ethyl 3-amino-4-(cyclohexylamino)benzoate (Fer-1) (100 mg,0.382 mmol, 1 equiv) and benzaldehyde (39 μL, 0.382 mmol, 1 equiv) wereheated in DCE for 1 hour at 80° C. in the presence of molecular sieve (4Å) then the mixture was cooled down to room temperature before additionof the NaBH(OAc)₃ in small portions over 3 hours. The reaction mixturewas stirred at room temperature under a nitrogen atmosphere for 17hours. The reaction mixture was quenched with aqueous saturated NaHCO₃,and the product was extracted with EtOAc. The EtOAc extract was dried(MgSO₄), and the solvent was evaporated. The residue was purified byflash-column chromatography on silica gel to provide the desired ethyl3-(benzylamino)-4-(cyclohexylamino)benzoate (SRS15-23, Scheme 3).

Method II: To ethyl 3-amino-4-(cyclohexylamino)benzoate (Fer-1) (100 mg,0.382 mmol, 1 equiv) and benzaldehyde (39 μL, 0.382 mmol, 1 equiv) inDCE was added NaBH(OAc)₃ (129.5 mg, 0.611 mmol, 1.6 equiv). The reactionmixture was treated in the same way as in method I.

General Procedure D (Alkylation Reaction)

A representative example is the methylation of the SRS15-24 using4-(bromomethyl)pyridine (Scheme 1). To the ethyl3-amino-4-(cyclooctylamino)benzoate (SRS15-18; 30 mg, 0.095 mmol) in THF(1 mL), 4-(bromomethyl)pyridine hydrogen bromide salt (24 mg, 0.095mmol) and DIPEA (50 μL, 0.285 mmol) were added. The mixture was stirredat 40° C. for 17 hours then poured in water. The organic layer wasextracted with EtOAc then dried under MgSO₄, and the solvent wasevaporated. The residue was purified by flash-column chromatography onsilica gel to provide the desired ethyl4-(adamanthylamino)-3-(pyridin-4-ylmethylamino)benzoate (SRS15-24).

General Procedure E (Imine Formation)

A representative example is the condensation reaction between SRS15-67and pyrimidine-5-carbaldehyde in ethanol in the presence of a catalyticamount of HCl (Scheme 3). To the ethyl3-amino-4-(adamantylamino)benzoate (SRS15-67; 100 mg, 0.318 mmol) inethanol (4 mL), drops of HCl (10 μL) were added. The mixture was stirredat 80° C. for 4 hours. The solvent was evaporated. The residue waspurified by flash-column chromatography on silica gel to provide thedesired (Z)-ethyl4-(adamantylamino)-3-(pyrimidin-5-ylmethyleneamino)benzoate (SRS15-72).

General Procedure F (Ester Formation)

A representative example is the esterification of4-chloro-3-nitrobenzoic acid using isopropanol or tert-butanol indichloromethane in the presence of N,N′-Dicyclohexylcarbodiimide (DCC)and 4-dimethylaminopyridine (DMAP) (Scheme 4). To the4-chloro-3-nitrobenzoic acid in dichloromethane was added4-dimethylaminopyridine and isopropanol. TheN,N′-Dicyclohexylcarbodiimide (DCC) was added, at 0° C., and the mixturewas stirred for 17 hours at room temperature. The precipitate wasfiltered out under celite and the organic layer was concentrated undervacuum. The residue was purified by flash-column chromatography onsilica gel to provide the desired ester (Scheme 4).

Synthesis of ethyl 3-amino-4-(1-adamantylamino)benzoate (SRS15-18.Scheme 1)

Following the above general procedure A and B and starting from thecommercially available ethyl 4-chloro-3-nitrobenzoate, the crudereaction mixture was purified by column chromatography (dichloromethane:methanol=40:1) to provide the desired ethyl3-amino-4-(1-adamantylamino)-benzoate (SRS15-18, Scheme 1) (649 mg,2.067 mmol, 95% (2 steps)). ¹H NMR (500 MHz, CDCl₃) δ 7.52 (d, J=8.1 Hz,1H), 7.43 (s, 1H), 6.93 (d, J=8.1 Hz, 1H), 4.34-4.27 (m, 2H), 2.15-1.72(m, 15H), 1.36 (t, J=6.6 Hz, 3H); LC/MS (APCI+, M+1) 315.36.

Synthesis of ethyl 3-(benzylamino)-4-(1-adamantylamino)benzoate(SRS15-23. Scheme 1)

Following the above general procedure C or D the crude reaction mixturewas purified by column chromatography (dichloromethane: methanol=100:1)to provide the desired ethyl3-(benzylamino)-4-(1-adamantylamino)benzoate (SRS15-23, Scheme 1) (26mg, 0.064 mmol, 68%). ¹H NMR (400 MHz, CDCl₃) δ 7.54 (dd, J=8.1, 1.5 Hz,1H), 7.49-7.27 (m, 7H), 6.98 (dd, J=11.3, 4.7 Hz, 1H), 4.37-4.28 (m,4H), 3.69 (s, 1H), 2.16 (s, 3H), 1.98 (s, 6H), 1.72 (s, 6H), 1.38 (ddd,J=7.1, 5.8, 1.6 Hz, 3H); LC/MS (APCI+, M+1) 405.36.

Synthesis of ethyl4-(1-adamantylamino)-3-((pyridin-4-ylmethyl)amino)-benzoate (SRS15-24.Scheme 1)

Following the above general procedure C or D the crude reaction mixturewas purified by column chromatography (dichloromethane: methanol=40:1)to provide the desired ethyl4-(1-adamantylamino)-3-((pyridin-4-ylmethyl)amino)-benzoate (SRS15-24,Scheme 1) (27 mg, 0.066 mmol, 70%). ¹H NMR (400 MHz, CDCl₃) δ 8.61 (s,2H), 7.53 (d, J=8.2 Hz, 1H), 7.38-7.26 (m, 4H), 6.99 (d, J=8.3 Hz, 1H),4.34 (dd, J=15.7, 8.6 Hz, 4H), 3.89 (s, 1H), 2.17-1.38 (m, 15H), 1.36(t, J=7.1 Hz, 4H); LC/MS (APCI+, M+1) 406.36.

Synthesis of ethyl4-(1-adamantylamino)-3-(pyrimidin-5-ylmethylamino)-benzoate (SRS15-25.Scheme 1)

Following the above general procedure C or D the crude reaction mixturewas purified by column chromatography (dichloromethane: methanol=40:1)to provide the desired ethyl4-(1-adamantylamino)-3-(pyrimidin-5-ylmethylamino)-benzoate (SRS15-25,Scheme 1) (23 mg, 0.056 mmol, 54%). ¹H NMR (400 MHz, CDCl₃) δ 9.24 (s,1H), 8.71 (t, J=5.0 Hz, 1H), 7.53 (d, J=8.3 Hz, 1H), 7.37 (d, J=4.9 Hz,1H), 7.31 (s, 1H), 7.00 (d, J=8.4 Hz, 1H), 4.47 (s, 2H), 4.33 (dd,J=9.6, 4.5 Hz, 2H), 2.17-1.40 (m, 15H), 1.36 (t, J=7.1 Hz, 3H); LC/MS(APCI+, M+1) 406.36.

Synthesis of ethyl 3-amino-4-(2-adamantylamino)benzoate (SRS15-17.Scheme 2)

Following the above general procedure A and B and starting from thecommercially available ethyl 4-chloro-3-nitrobenzoate (Scheme 2), thecrude reaction mixture was purified by column chromatography(dichloromethane: methanol=40:1) to provide the desired ethyl3-amino-4-(2-adamantylamino)-benzoate (SRS15-17, Scheme 2) (624 mg, 1.98mmol, 91% (2 steps)). ¹H NMR (500 MHz, CDCl₃) δ 7.60 (s, 1H), 7.45 (s,1H), 6.57 (s, 1H), 4.86 (s, 1H), 4.34 (d, J=5.4 Hz, 2H), 3.83 (s, 1H),3.65 (s, 1H), 3.29 (s, 1H), 2.04-1.60 (m, 14H), 1.38 (d, J=3.8 Hz, 3H);LC/MS (APCI+, M+1) 315.36.

Synthesis of ethyl 3-(benzylamino)-4-(2-adamantylamino)benzoate(SRS15-20 mono. Scheme 2)

Following the above general alkylation reaction (procedure C) the crudereaction mixture was purified by column chromatography (dichloromethane:methanol=100:1) to provide the desired ethyl 3-(benzylamino)-4-(2adamantylamino)-benzoate (SRS15-20_mono, Scheme 2) (19 mg, 0.047 mmol,50%) and the dialkylation compound, the ethyl3-(dibenzylamino)-4-(2-adamantylamino)-benzoate, (SRS15-20_di, Scheme 2)(9 mg, 0.018 mmol, 19%). ¹H NMR (400 MHz, CDCl₃) δ 7.68-7.61 (m, 1H),7.52 (d, J=1.7 Hz, 1H), 7.49-7.25 (m, 5H), 6.69-6.52 (m, 1H), 4.53 (s,1H), 4.40-4.29 (m, 4H), 3.67 (s, 1H), 3.23 (s, 1H), 2.09 (s, 2H),1.99-1.86 (m, 8H), 1.80 (s, 2H), 1.66-1.58 (m, 2H), 1.41-1.36 (m, 3H);LC/MS (APCI+, M+1) 405.36.

Synthesis of ethyl 3-(dibenzylamino)-4-(2-adamantylamino)benzoate(SRS15-20 di. Scheme 2)

¹H NMR (400 MHz, CDCl₃) δ 7.80 (s, 1H), 7.72 (d, J=8.5 Hz, 1H), 7.28(dd, J=9.0, 1.4 Hz, 10H), 6.46 (d, J=8.6 Hz, 1H), 5.99 (d, J=7.0 Hz,1H), 4.38-4.28 (m, 2H), 4.06 (s, 4H), 3.54 (s, 1H), 2.00-1.50 (m, 14H),1.39 (t, J=7.1 Hz, 3H); LC/MS (APCI+, M+1) 495.36.

Synthesis of ethyl4-(2-adamantylamino)-3-((pyridin-4-ylmethyl)amino)-benzoate (SRS15-21.Scheme 2)

Following the above general procedure C or D the crude reaction mixturewas purified by column chromatography (dichloromethane: methanol=40:1)to provide the desired ethyl4-(2-adamantylamino)-3-((pyridin-4-ylmethyl)amino)-benzoate (SRS15-21,Scheme 2) (25 mg, 0.061 mmol, 65%). ¹H NMR (400 MHz, CDCl₃) δ 8.62 (s,2H), 7.64 (d, J=8.2 Hz, 1H), 7.46-7.31 (m, 3H), 6.63 (d, J=8.4 Hz, 1H),4.41 (s, 2H), 4.35-4.25 (m, 2H), 3.68 (s, 1H), 2.09 (s, 2H), 1.94 (d,J=15.1 Hz, 8H), 1.81 (s, 2H), 1.72-1.64 (m, 2H), 1.36 (t, J=7.1 Hz, 3H);LC/MS (APCI+, M+1) 406.26.

Synthesis of ethyl4-(2-adamantylamino)-3-(pyrimidin-5-ylmethylamino)-benzoate (SRS15-22.Scheme 2)

Following the above general procedure C or D the crude reaction mixturewas purified by column chromatography (dichloromethane: methanol=40:1)to provide the desired ethyl4-(2-adamantylamino)-3-(pyrimidin-5-ylmethylamino)-benzoate (SRS15-22,Scheme 2) (26 mg, 0.064 mmol, 60%). ¹H NMR (400 MHz, CDCl₃) δ 9.24 (s,1H), 8.71 (d, J=5.1 Hz, 1H), 7.69-7.59 (m, 1H), 7.38 (d, J=2.0 Hz, 2H),6.63 (d, J=8.4 Hz, 1H), 4.47 (d, J=21.3 Hz, 2H), 4.32 (dd, J=14.2, 7.1Hz, 2H), 4.15 (s, 1H), 3.69 (s, 1H), 2.04-1.60 (m, 14H), 1.36 (d, J=7.1Hz, 3H); LC/MS (APCI+, M+1) 407.46.

Synthesis of ethyl4-(adamantylamino)-3-(pyrimidin-5-ylmethyleneamino)benzoate (SRS15-72,Scheme 2)

Following the above general procedure E, the reaction was purified byflash-column chromatography on silica gel to provide the desired ethyl4-(adamantylamino)-3-(pyrimidin-5-ylmethyleneamino)benzoate (SRS15-72,Scheme 3) (99 mg, 0.245 mmol, 77%). ¹H NMR (400 MHz, CDCl₃) δ 9.31 (s,1H), 9.22 (s, 2H), 8.68 (s, 1H), 7.89-7.78 (m, 2H), 7.00 (d, J=8.7 Hz,1H), 5.62 (s, 1H), 4.37 (q, J=7.1 Hz, 2H), 2.20 (s, 3H), 2.09 (d, J=2.5Hz, 6H), 1.77 (s, 6H), 1.40 (t, J=7.1 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃)δ 166.72, 159.78, 156.48, 150.74, 146.56, 134.47, 130.69, 129.70,117.92, 117.03, 112.46, 60.38, 51.89, 42.52, 36.38, 29.60, 14.49; LC/MS(APCI+, M+1) 405.57.

Synthesis of ethyl4-(adamantylamino)-3-(pyrimidin-5-ylmethylamino)benzoate (SRS16-41,Scheme 2)

Following the above general procedure C, the reaction was purified byflash-column chromatography on silica gel to provide the desired ethyl4-(adamantylamino)-3-(pyrimidin-5-ylmethylamino)benzoate (SRS16-41,Scheme 3) (80 mg, 0.197 mmol, 80%). ¹H NMR (400 MHz, CDCl₃) δ ¹H NMR(400 MHz, CDCl₃) δ 9.17 (s, 1H), 8.77 (s, 2H), 7.55 (d, J=8.3 Hz, 1H),7.40 (s, 1H), 6.96 (d, J=8.3 Hz, 1H), 4.37-4.29 (m, 4H), 3.76 (s, 1H),2.14 (s, 3H), 1.94 (s, 6H), 1.72 (d, J=12.3 Hz, 6H), 1.35 (dd, J=9.3,4.9 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 166.93, 157.98, 156.52, 140.64,136.71, 132.54, 123.24, 120.65, 116.53, 115.76, 60.37, 52.51, 44.83,42.88, 36.40, 29.63, 14.43; LC/MS (APCI+, M+1) 407.27.

Following the above general procedure F, A and B respectively andstarting from the commercially available 4-chloro-3-nitrobenzoic acid(Scheme 4), the crude reaction mixture was purified by columnchromatography (dichloromethane: methanol=40:1) to provide the desiredisopropyl 3-amino-4-(2-adamantylamino)-benzoate (SRS16-78, Scheme 4)(600 mg, 1.83 mmol, 95% (3 steps). ¹H NMR (400 MHz, CDCl₃) δ 7.50 (d,J=6.4 Hz, 1H), 7.41 (s, 1H), 6.92 (d, J=7.2 Hz, 1H), 5.19 (s, 1H), 3.36(s, 2H), 2.12 (s, 3H), 1.96 (s, 6H), 1.70 (s, 6H), 1.33 (s, 6H); LC/MS(APCI+, M+1) 329.06.

Following the above general procedure F, A and B respectively andstarting from the commercially available 4-chloro-3-nitrobenzoic acid(Scheme 4), the crude reaction mixture was purified by columnchromatography (dichloromethane: methanol=40:1) to provide the desiredtert-butyl 3-amino-4-(2-adamantylamino)-benzoate (SRS16-82, Scheme 4)(700 mg, 2.046 mmol, 88% (3 steps). ¹H NMR (400 MHz, CDCl₃) δ 7.47 (dd,J=8.4, 1.9 Hz, 1H), 7.38 (d, J=1.8 Hz, 1H), 6.93 (d, J=8.4 Hz, 1H), 3.33(s, 2H), 2.15 (s, 3H), 1.99 (s, 6H), 1.72 (s, 6H), 1.57 (s, 9H); LC/MS(APCI+, M+1) 343.09.

Following the above general procedure E, the reaction was purified byflash-column chromatography on silica gel to provide the desiredisopropyl 4-(adamantylamino)-3-(pyrimidin-5-ylmethyleneamino)benzoate(SRS15-80, Scheme 4) (90 mg, 0.215 mmol, 77%). ¹H NMR (400 MHz, CDCl₃) δ9.40-9.03 (m, 3H), 8.68 (d, J=5.8 Hz, 1H), 7.81 (dd, J=21.3, 5.2 Hz,2H), 7.29 (d, J=5.7 Hz, 1H), 7.00 (d, J=8.7 Hz, 1H), 5.61 (s, 1H), 5.25(dd, J=12.5, 6.3 Hz, 1H), 2.19 (s, 3H), 2.08 (s, 6H), 1.76 (s, 6H), 1.37(d, J=6.2 Hz, 6H); LRMS (FAB+, M+) 419.3; HRMS (FAB+) calculated forC₁₆H₂₄N₂O₂, 418.2369; found: 418.2365.

Following the above general procedure E, the reaction was purified byflash-column chromatography on silica gel to provide the desiredtert-butyl 4-(adamantylamino)-3-(pyrimidin-5-ylmethyleneamino)benzoate(SRS15-86, Scheme 4) (200 mg, 0.462 mmol, 89%). ¹H NMR (400 MHz, CDCl₃)δ 9.30 (s, 10H), 9.29-9.15 (m, 33H), 8.66 (d, J=4.1 Hz, 14H), 7.99-7.70(m, 28H), 7.19-6.92 (m, 14H), 5.58 (s, 13H), 2.13 (d, J=46.0 Hz, 117H),2.00 (s, 21H), 1.75 (s, 106H), 1.71-1.54 (m, 140H); LRMS (FAB+, M+)432.1; HRMS (FAB+) calculated for C₁₆H₂₄N₂O₂, 432.2525; found: 432.2541.

Additional Ferrostatin-1 Analogs

The following Ferrostatin-1 analogs may be made according to the generalprocedures set forth above:

Example 3 Erastin Triggers Oxidative, Iron-Dependent Cell Death

RSL-induced cell death is a poorly characterized process involving theaccumulation of ROS derived from an unknown source and the inhibition ofcell death by iron chelation (Yagoda et al., 2007; Yang and Stockwell,2008). It was observed that these two processes were linked. Treatmentof NRAS-mutant HT-1080 fibrosarcoma cells with the RSL molecule erastin(10 μM) resulted in a time-dependent increase in cytosolic and lipid ROSbeginning at 2 hours, as assayed by flow cytometry using the fluorescentprobes H₂DCFDA and C11-BODIPY, respectively (FIGS. 1B and 1C). Thisincrease in ROS preceded cell detachment and overt death, which began at6 hours (FIG. 1A). ROS accumulation and cell death were suppressed byco-treatment with the iron chelator deferoxamine (DFO, 100 μM) (FIGS.1A-C), while incubation with three different exogenous sources of iron,but not by other divalent transition metal ions (Cu²⁺, Mn²⁺, Ni²⁺,Co²⁺), potentiated erastin-induced death (FIGS. 8A and 8B). Because celldeath occurred in erastin-treated cells following a prolonged period ofROS accumulation and was suppressed by antioxidants (see below), thedata suggest that the overwhelming, iron-dependent accumulation of ROSis what kills these cells.

Because two erastin targets, VDAC2 and VDAC3, reside in themitochondria, it was hypothesized that erastin-induced death involvedaberrant ROS production by the mitochondrial electron transport chain(ETC). However, in erastin-treated (10 μM, 6 hours) HT-1080 cells, noincrease in MitoSOX-sensitive mitochondrial ROS production was observed(FIG. 1D, left). The ETC complex I inhibitor rotenone (250 nM, 6 hours)enhanced MitoSOX-sensitive ROS production, but in a manner that wasinsensitive to DFO (FIG. 1D, right). Furthermore, KRAS-mutant 143Bosteosarcoma cells incapable of ETC-dependent ROS formation, due to thedepletion of mitochondrial DNA (mtDNA)-encoded transcripts (ρ⁰ cells),were as sensitive to erastin and RSL3 as matched mtDNA-wild-type (ρ⁺)cells (FIGS. 1E, 1F, and 8C-E). Thus, erastin-induced cell death inhuman cancer cells involves DFO-sensitive ROS accumulation and can occurin cells lacking a functional ETC. This iron-dependent death phenotypewas named ferroptosis.

Example 4 Ferroptosis is Distinct from Known Forms of Cell Death

Whether ferroptosis shared morphological, bioenergetic or othersimilarities with apoptotic or necrotic death, or with autophagy wasexamined. Using transmission electron microscopy, it was observed thatHRAS-mutant BJeLR engineered tumor cells treated with erastin exhibitednone of the characteristic morphologic features associated withstaurosporine (STS)-induced apoptosis (e.g. chromatin condensation andmargination), hydrogen peroxide (H₂O₂)-induced necrosis (e.g.cytoplasmic and organelle swelling, plasma membrane rupture) orrapamycin-induced autophagy (e.g. formation of double-membrane enclosedvesicles) (FIG. 2A). The lone distinctive morphological feature oferastin-treated cells were mitochondria that appeared smaller thannormal, with increased membrane density, consistent with the previousreport (Yagoda et al., 2007) (FIG. 2A). With respect to bioenergetics,substantial depletion of intracellular ATP in BJeLR and HT-1080 cellstreated with H₂O₂, but not erastin, STS or rapamycin, was observed (FIG.2B), thus distinguishing ferroptosis from various forms of necrosis thatinvolve bioenergetic failure.

Using a variation of the modulatory profiling strategy (Wolpaw et al.,2011), the ability of twelve established small molecule cell deathinhibitors to prevent ferroptosis in HT-1080, BJeLR and KRAS-mutantCalu-1 non-small cell lung cancer cells was tested. The modulatoryeffect (M_(e)) for each inhibitor (tested in a 10-point, 4-fold dilutionseries) on the normalized viability of cells treated with a lethal doseof erastin (M_(e)<0: death sensitization; M_(e)=0: no effect; M_(e)>0:death rescue) was computed. The resultant values were clusteredhierarchically in an unsupervised fashion and displayed as a heatmap.Using this approach, it was observed that erastin-induced death was notconsistently modulated by inhibitors of caspase, cathepsin or calpainproteases (z-VAD-fmk, E64d or ALLN), RIPK1 (necrostatin-1), cyclophilinD (cyclosporin A) or lysosomal function/autophagy (bafilomycin A1,3-methyladenine, chloroquine), compounds known to inhibit various formsof apoptosis, necrosis and autophagic cell death (FIG. 2C).

DFO, the anti-oxidant trolox, the MEK inhibitor U0126 and, to a weakerextent, the protein synthesis inhibitor cycloheximide (CHX), all rescuedfrom erastin-induced death in HT-1080, BJeLR and Calu-1 cells (FIG. 2C)(Yagoda et al., 2007). These inhibitors were also effective atpreventing erastin-induced ferroptosis in both wild-type andapoptosis-deficient Bax/Bak double knockout (DKO) mouse embryonicfibroblasts (FIGS. 9A and 9B), indicating that ferroptosis can beactivated in human- and mouse-derived cells and is independent of thecore apoptotic machinery regulated by Bax and Bak. DFO, trolox and U0126all prevented the accumulation of H₂DCFDA-sensitive ROS inerastin-treated HT-1080 cells (FIG. 2D), demonstrating that theseinhibitors act to prevent death upstream or at the level of ROSproduction. Because trolox, U0126 and the membrane permeable ironchelator 2,2-bipyridyl could be added to HT-1080 cells up to 6 hoursafter erastin and still confer substantial protection from death (FIG.9C), ferroptosis likely requires continuous iron-dependent ROS formationover an extended period of time to trigger death.

Finally, in a modulatory profiling experiment that tested the ability ofDFO, trolox, U0126, CHX, the membrane permeable iron chelator ciclopiroxolamine (CPX) and the glutathione peroxidase mimetic ebselen (Ebs) tomodulate the lethality of erastin, RSL3 or sixteen other mechanisticallydistinct lethal compounds thought to kill cells through variousROS-dependent and -independent mechanisms, it was observed that erastinand RSL3 formed a distinct cluster, separate from all other inducers ofcell death (FIG. 2E). Together, these data support the hypothesis thatRSL-induced ferroptosis is a novel death phenotype distinct fromapoptosis, various forms of necrosis and autophagy.

Example 5 Ferroptosis is Regulated by a Distinct Set of Genes

To explore the genetic basis of ferroptosis, genes uniquely required forthis process were identify. The potential role of the mitochondria werefocused on, because this organelle displayed an aberrant morphology inerastin-treated cells (FIG. 2A). Mitochondrial gene function wasperturbed using a custom arrayed shRNA library targeting 1,087 genes(median 5 hairpins/gene), most of which (901, 88%) encode predictedmitochondrial proteins (Pagliarini et al., 2008) (FIG. 3A). Using thislibrary, the genetic suppressibility of erastin (7.3 μM)-inducedferroptosis and STS (1 μM)-induced apoptosis in Calu-1 cells wascompared (FIG. 3A). Across all 5,817 informative hairpins, nosignificant correlation between those shRNAs that rescued fromerastin-induced ferroptosis and from STS-induced apoptosis (Spearmanrank sum test, r=−0.01, P=0.46) was observed, thus confirming thatdistinct genetic networks govern erastin-induced ferroptosis andSTS-induced apoptosis.

Next, a second erastin resistance screen in HT-1080 cells was performedand, using a rigorous confirmation pipeline, six high-confidence geneswere identified. These six high-confidence genes were supported by atleast two independent shRNAs per gene that are required forerastin-induced ferroptosis in both HT-1080 and Calu-1 cells—RPL8(ribosomal protein L8), IREB2 (iron response element binding protein 2),ATP5G3 (ATP synthase F₀ complex subunit C3), CS (citrate synthase),TTC35 (tetratricopeptide repeat domain 35) and ACSF2 (acyl-CoAsynthetase family member 2) (FIGS. 3B and 3C). Consistent with theestablished CHX- and DFO-sensitive nature of erastin-inducedferroptosis, RPL8 encodes a component of the 60S ribosomal subunitpresumably required for translation and IREB2 encodes a master regulatorof iron metabolism. These results were further validated. It was foundthat shRNA-mediated silencing of IREB2 and the IREB2 negative regulatorFBXL5 (Salahudeen et al., 2009; Vashisht et al., 2009) resulted inreciprocal changes in the expression of the known iron uptake,metabolism and storage genes TFRC, ISCU, FTH1, FTL and in erastinsensitivity (FIG. 10A-C). These results provide confidence in thequality of the screening and confirmation procedures.

To establish the generalizability of the results obtained in HT-1080 andCalu-1 cells, the effects of silencing these genes in HT-1080, Calu-1and six additional cell lines treated with erastin were tested.Silencing of these six high confidence genes using the most effectivehairpin for each gene, defined by mRNA silencing levels in HT-1080 cells(FIG. 3C), conferred ≥20% rescue in 79% (38/48) of shRNA-cell linecombinations (FIG. 3D). Thus, these genes appear to be broadly requiredfor erastin-induced ferroptosis. Next, whether silencing of these genesspecifically attenuated erastin-induced ferroptosis, or more broadlymodulated a variety of lethal effects was tested. Silencing of these sixgenes conferred protection against erastin-induced ferroptosis (≥40%rescue for 6/6 hairpins), but not cell death/cytostasis induced by STS,rotenone, rapamycin, the proteasome inhibitor MG132, the DNA-damagingagent camptothecin or the Ca²⁺-dependent ATPase inhibitor thapsigargin(≥40% rescue for 0/6 hairpins) (FIG. 3E). Together, these data supportthe hypothesis that a unique genetic network governs erastin-inducedferroptosis compared to other forms of cell death.

Both ACSF2 and CS are implicated in the regulation of mitochondrialfatty acid metabolism (Mullen et al., 2011; Watkins et al., 2007).Whether this process could contribute to ferroptosis was examined. Incancer cells, fatty acid synthesis is in part dependent upon themetabolism of glutamine (Gln) to alphaketoglutarate, a process that canbe inhibited by the small molecule transaminase inhibitor aminooxyaceticacid (AOA) (Wise et al., 2008) (FIG. 3F). In cell culture mediacontaining both glucose and Gln, AOA (2 mM) rescued both HT-1080 andBJeLR cells from erastin-induced ferroptosis (FIGS. 3F, 10D), mimickingthe effects of silencing CS and ACSF2. In AOA-treated HT-1080 cells, thelethality of erastin was restored by co-incubation with dimethyl alphaketoglutarate (DMK), which provides the downstream metabolite whoseproduction from Gln is blocked by AOA (Wise et al., 2008) (FIGS. 3F and3G). An unrelated modulator of mitochondrial function not predicted todirectly affect Gln metabolism, dichloroacetic acid (DCA), had no effecton erastin-induced ferroptosis (FIG. 10D). These results suggest that aGln-CS- and ACSF2-dependent lipid synthesis pathway could supply aspecific lipid precursor required for ferroptosis.

Example 6 Identification of Ferrostatin-1 as a Small Molecule Inhibitorof Ferroptosis

One ultimate aim is to investigate the potential role of ferroptosis invivo. Therefore, a potent and specific drug-like small moleculeinhibitor of this process was identified. As set forth above, toovercome the inherent limitations of many individual small moleculecollections (Macarron et al., 2011), a custom screening library of 9,517small molecules derived from a starting pool of 3,372,615 commerciallyavailable compounds that were filtered in silico on the basis ofdrug-likeness, solubility, scaffold diversity and other parameters wasassembled. Screening of this lead-optimized compound′ (LOC) library andsubsequent confirmation studies identified a compound, which theinventors named ferrostatin-1 (Fer-1), as the most potent inhibitor oferastin-induced ferroptosis in HT-1080 cells (EC₅₀=60 nM) (FIGS. 4A,10A, and 10B). To the inventors' knowledge, the activity for Fer-1 hasnot previously been reported in any biological system. A total synthesisof Fer-1 was performed as set forth above, and this material was used toconfirm the activity of Fer-1 and to demonstrate that it specificallyinhibited RSL-induced death, but not cell death induced by otheroxidative lethal compounds and apoptosis-inducing agents (FIGS. 4B, 6A).

The Fer-1 mechanism of action was examined. Fer-1 did not inhibit ERKphosphorylation or arrest the proliferation of HT-1080 cells, suggestingthat it does not inhibit the MEK/ERK pathway, chelate iron or inhibitprotein synthesis (FIGS. 4C and 4D). Fer-1 did, however, preventerastin-induced accumulation of cytosolic and lipid ROS (FIG. 4E).Moreover, similar to the positive control antioxidant trolox, Fer-1readily oxidized the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH)under cell free conditions, a test of intrinsic antioxidant potential(FIG. 4F). Substitution of the primary aromatic amine for a nitro group(SRS8-24), or elimination of the N-cyclohexyl moiety (CA-1), destroyedthe antioxidant capability of Fer-1, as well as its ability to preventerastin (10 μM)-induced death in HT-1080 cells (FIGS. 4F-H). Thus, botharomatic amines are required for Fer-1 to prevent RSL-induced death, afunction plausibly linked to its ability to scavenge free radicals.

The results suggested that lipid ROS were crucial for erastin-induceddeath. The inventors therefore hypothesized that Fer-1 was a lipid ROSscavenger, with the N-cyclohexyl moiety serving as a lipophilic anchorwithin biological membranes. Consistent with this hypothesis, in aseries of ten Fer-1 analogs, where the number of carbons in theN-substituted cyclic moiety was systematically varied, a significantcorrelation between the predicted lipophilicity (octanol-water partitioncoefficient, log P) and the erastin-death-suppressing ability of eachmolecule (Spearman R=−0.85, P=0.002) was observed (FIGS. 4I and 11C). Ofnote, SRS8-72, a Fer-1 analog with N-cyclopropyl in place ofN-cyclohexyl, which was an order of magnitude less potent than Fer-1 atpreventing death, nonetheless retained equivalent intrinsic antioxidantcapability in the cell-free DPPH assay (FIGS. 4F-H and 10C). Thus, theN-cyclohexyl moiety likely enables Fer-1 to prevent ferroptosis bypromoting the tethering of Fer-1 within lipid membranes, as opposed toinfluencing the intrinsic antioxidant potential of this molecule.

Intriguingly, lipid partitioning alone does not appear to be sufficientto account for the potency of Fer-1. Fer-1 has similar predictedlipophilicity, but much greater erastin-suppressing potency than twocanonical lipophilic antioxidants (trolox and butylated hydroxyltoluene[BHT]), while being both considerably more lipophilic and more potentthan two representative soluble antioxidants (Tiron, TEMPO) (FIGS. 4Jand 4K). Both trolox and BHT are phenolic antioxidants, while Fer-1contains an aromatic amine. It was hypothesized that this difference mayconfer a unique profile of radical reactivity upon Fer-1 that is bettertuned to the RSL mechanism.

Example 7 Fer-1 Prevents Glutamate-Induced Neurotoxicity

Excitotoxic cell death that occurs in the nervous system in epilepsy,stroke and other trauma situations has also been described as anoxidative, iron-dependent process (Cheah et al., 2006; Choi, 1988;Murphy et al., 1989). It was hypothesized that excitotoxic death couldbe related to erastin-induced ferroptosis. This hypothesis was testedusing a rat organotypic hippocampal slice culture (OHSC) model thatclosely resembles the hippocampus in vivo by preserving the integrity ofneuronal connections, both inhibitory and excitatory, and theirsupporting cells, including astrocytes and microglia (Lossi et al.,2009). OHSCs have proven to be ideal complex preparations forlead-compound identification and validation (Noraberg et al., 2005;Sundstrom et al., 2005), capable of predicting in vivo efficacy (Cateret al., 2007; Morrison et al., 2002).

OHSCs were treated with a lethal excitotoxic stimulus (5 mM L-glutamate,3 hours) that mimics the consequences of stroke and neurodegenerativedisease (Morrison et al., 2002; Sundstrom et al., 2005) (FIG. 5A). Theseslices were co-incubated with glutamate and vehicle alone or withglutamate plus Fer-1 (2 μM), the iron chelator CPX (5 μM) or, as apositive control, the NMDA receptor antagonist MK-801 (10 μM). Theeffects of these compound treatments on propidium iodide (PI) uptakewere analyzed, as an indicator of cell death, 24 hours following the endof glutamate treatment, in 3 defined regions of the OHSCs: the dentategyrus (DG), the CA1 and the CA3 fields of the hippocampus. A two-wayanalysis of variance (ANOVA) suggested significant differences for bothbrain region (F_(2,75)=19.23, P<0.0001) and compound treatment(F_(4,75)=67.8, P<0.0001) factors. Focusing on the compound treatmenteffect, Bonferroni post-tests indicated that glutamate inducedsignificant cell death in all three regions of the brain, and that thisdeath was attenuated significantly and to an almost identical extent byco-treatment with Fer-1, CPX or MK-801 (P<0.001 for all interactionsexcept glutamate+MK-801 within the DG, P<0.01) (FIG. 5B-E). Theseresults suggest that glutamate-induced death in OHSCs anderastin-induced death in cancer cells share in common a core lethalmechanism that can be inhibited by iron chelation or Fer-1.

Example 8 Erastin Inhibits System x_(c) ⁻

CPX and Fer-1 suppressed erastin-induced death in cancer cells andglutamate-induced toxicity in OHSCs, consistent with a common iron- andROS-dependent death execution mechanism. Whether any death-initiatingmechanisms could also be shared between these two processes wasinvestigated.

Glutamate-induced death in brain cells can be initiated by calciuminflux through ionotropic glutamate receptors and through competitiveinhibition of cystine uptake by the Na⁺-independent cystine/glutamateantiporter, system x_(c) ⁻ (Choi, 1988; Murphy et al., 1989). Thecalcium chelators BAPTA-AM, Fura-2 and EGTA had no effect onerastin-induced death (FIG. 12A) (Wolpaw et al., 2011), arguing againsta role for Ca²⁺ influx in this process. However, striking clustering oferastin and sulfasalazine (SAS), a specific inhibitor of system x_(c) ⁻(Gout et al., 2001), was observed in a modulatory profile of 19oxidative and non-oxidative lethal molecules generated in HT-1080 cells(FIG. 6A). If blockade of system x_(c) ⁻-mediated cystine import cantrigger ferroptosis, then providing this metabolite to cells through analternative means should rescue from death. Indeed, β-mercaptoethanol(β-ME), which can circumvent the inhibition of system x_(c) ⁻ bypromoting cystine uptake through an alternative pathway (Ishii et al.,1981), strongly inhibited cell death in HT-1080 cells induced byerastin, SAS and glutamate (FIGS. 6A and 12B). As predicted by theseresults, SAS, like erastin, behaved as an oncogenic RAS-selective lethal(RSL) compound, albeit with considerably lower potency than erastin(FIG. 12C). This is nonetheless noteworthy, as SAS is an FDA-approveddrug not previously shown to demonstrate such activity.

System x_(c) ⁻ is a disulfide-linked heterodimer composed of SLC7A11(xCT) and SLC3A2 (4F2hc, CD98hc) (Sato et al., 1999) (FIG. 6B).Inhibition of system x_(c) ⁻ can lead to a compensatory transcriptionalup-regulation of SLC7A11 (Lo et al., 2008). Consistent with this,substantial upregulation of SLC7A11 in HT-1080 cells treated witherastin or SAS was observed. This effect was suppressed by β-ME, but notDFO or Fer-1 (FIG. 6C). Further confirming the relevance of system x_(c)⁻ to erastin-induced ferroptosis, siRNA-mediated silencing of SLC7A11with two independent siRNAs sensitized HT-1080 cells to erastin-induceddeath (FIGS. 6D and 6E), while transfection of HT-1080 cells with aplasmid encoding DDK-tagged SLC7A11 conferred protection from erastin-and SAS-induced death (FIG. 12D). Given these results, the uptake of[¹⁴C]cystine in HT-1080 cells was directly examined. Erastin (10 μM),glutamate (50 mM) and SAS (1 mM) abolished the Na⁺-independent uptake of[¹⁴C]cystine while RSL3 had no effect on this process (FIGS. 6F, 12E).

How erastin inhibits system x_(c) ⁻ was investigated. Analysis ofaffinity purification data (Yagoda et al., 2007) identified SLC7A5(LAT1, 4F2lc, CD98lc) as the lone protein bound by an active erastinaffinity analog in lysates from both HRAS-wildtype BJeH and HRAS-mutantBJeLR cells (FIG. 6G). SLC7A5 (like SLC7A11) is one of six light chainsthat bind SLC3A2 to form amino acid transporters of differing substrateselectivity. The SLC7A5/SLC3A2 complex (system L) transports large,neutral amino acids (Kanai and Endou, 2003) (FIG. 6B). In a profile of123 metabolites from human Jurkat T lymphocytes treated with erastin (1μM, 25 min) (Ramanathan and Schreiber, 2009), highly significantdecreases were observed in the levels of system L substrates (Kanai andEndou, 2003), while the levels of non-system L substrates were unchangedor increased (FIG. 6H). However, unlike inhibition of system x_(c) ⁻using excess glutamate (12.5 mM), inhibition of system L using excessD-phenylalanine (12.5 mM) (Kanai and Endou, 2003) did not stronglysensitize to erastin (FIG. 6I). Together, these results suggest thaterastin inhibits system L-mediated amino acid uptake, but that this doesnot contribute directly to ferroptosis. Rather, erastin binding toSLC7A5 or the SLC7A5/SLC3A2 complex interferes with cystine uptake bythe SLC3A2/SLC7A11 complex in trans.

Example 9 NAPDH Oxidases Provide One Source of Death-Inducing Ros inErastin-Treated Cells

Blocking system x_(c) ⁻ inhibits cysteine-dependent glutathione (GSH)synthesis and inhibits the trans-plasma membrane cysteine redox shuttle(Banjac et al., 2008; Ishii et al., 1981). Both effects impair cellularantioxidant defenses, thereby facilitating toxic ROS accumulation.Having ruled out the mitochondrial ETC as a source of death-inducing ROSin erastin-treated cells (FIGS. 1D-F), the role of the NADPH oxidase(NOX) family of superoxide-producing enzymes (NOX1-5, DUOX1,2), whichare up-regulated in several RAS-mutant tumors (Kamata, 2009) wasexamined. Erastin-induced ferroptosis was strongly suppressed in Calu-1cells by the canonical NOX inhibitor diphenylene iodonium (DPI), theNOX1/4 specific inhibitor GKT137831 (Laleu et al., 2010) and aninhibitor of the NADPH-generating pentose phosphate pathway (PPP),6-aminonicotinamde (6-AN) (FIGS. 7A and 7B). Given that Calu-1 cellsexpress NOX1 at much higher levels than NOX4 (FIG. 13A), NOX1 is themost likely candidate to mediate the observed NOX-dependent lethaleffects in these cells. Additionally, shRNA-mediated silencing of twoPPP enzymes, glucose-6-phosphate dehydrogenase (G6PD) andphosphoglycerate dehydrogenase (PGD), also prevented erastin-inducedferroptosis in Calu-1 cells to the same extent as silencing of VDAC2(FIGS. 7C and 7D). 6-AN also prevented cell death as well as ROSproduction in BJeLR cells (FIGS. 13B and 13C), suggesting an importantrole for this pathway is these cell types. On the other hand, NOX andPPP inhibitors were only partially effective at preventingerastin-induced ferroptosis in HT-1080 cells (FIG. 7B), indicating thatother pathways, in addition to the PPP/NOX pathway, can contribute tothe onset of death in erastin-treated cells, once the appropriateconditions have been set by the inhibition of system x_(c) ⁻.

Ferroptotic death is morphologically, biochemically and geneticallydistinct from apoptosis, various forms of necrosis, and autophagy. Thisprocess is characterized by the overwhelming, iron-dependentaccumulation of lethal lipid ROS (FIG. 7E, blue outline). Unlike otherforms of apoptotic and non-apoptotic death (Christofferson and Yuan,2010; Jacobson and Raff, 1995), this requirement for ROS accumulationappears to be universal. In at least some cells, NOX-family enzymes makeimportant contributions to this process. Indeed, although thepossibility of a death-inducing protein or protein complex activateddownstream of ROS accumulation cannot be excluded, the inventors positthat the executioners of death in cancer cells undergoing ferroptosisare these ROS themselves. An important prediction of this model is thatunder anoxic conditions ferroptosis will be inactive. However, evenhere, agents such as erastin that prevent uptake of essential aminoacids by system L are likely to be toxic to cells.

Using an shRNA library targeting most known genes encoding mitochondrialproteins (Pagliarini et al., 2008), specific roles for RPL8, IREB2,ATP5G3, TTC35, CS and ACSF2 in erastin-induced ferroptosis wereidentified. A plausible new hypothesis to emerge from these data is thatCS and ACSF2 are required to synthesize a specific lipid precursornecessary for death (FIG. 7E). Just as important, the high-resolution ofthe arrayed approach (1 hairpin/well, minimum 5 hairpins/gene) providesconfidence that the various mitochondrial genes not identified in thescreen, including many implicated in apoptotic and non-apoptotic death(BID, BAK1, BAX, AIFM1, PP/F, HTRA2, ENDOG, PGAM5), are truly notrequired for erastin-induced ferroptosis. This screening collection willbe a valuable resource for future studies of the role of themitochondria in cell physiology.

In cancer cells, inhibition of system x_(c) ⁻-mediated cystine uptake byerastin, SAS or glutamate may be sufficient to initiate iron-dependentferroptosis. Inhibition of system x_(c) ⁻ is, however, not necessary:RSL3 does not inhibit cystine uptake and yet triggers an otherwisesimilar iron and ROS-dependent ferroptototic death program. Thus, RSL3likely modulates the activity of a target lying downstream of or inparallel to system x_(c) ⁻ (FIG. 7E). Importantly, this may enable RSL3to activate ferroptosis in cells or conditions where cystine uptake viasystem x_(c) ⁻ is not limiting for survival. Lanperisone, anotherrecently identified oncogenic RAS-selective lethal small molecule thatcauses non-apoptotic, iron-dependent death in mouse Kras-mutant tumorcells (Shaw et al., 2011), may also inhibit the function of system x_(c)⁻ or another target in the ferroptotic pathway. Other compounds thatbehave as RSLs, such as PEITC, oncrasin and piperlongumine (Guo et al.,2008; Raj et al., 2011; Trachootham et al., 2006), trigger mitochondrialcytochrome C release, caspase activation and other features of apoptosisnot observed in cancer cells undergoing ferroptosis. Certain tumor cellsare highly resistant to apoptosis (Ni Chonghaile et al., 2011). Thus,agents such as erastin, RSL3 and lanperisone that can triggernon-apoptotic death may exhibit a unique spectrum of clinical activity.

In some brain cell populations, inhibition of system x_(c) ⁻ byglutamate triggers oxidative cell death dependent on iron and lipid ROS,but also Ca²⁺ influx, mitochondrial damage, mitochondrial ROS productionand chromatin fragmentation (Li et al., 1997; Murphy et al., 1989; Ratanet al., 1994; Tan et al., 1998; Yonezawa et al., 1996). These latterevents are not required for RSL-induced ferroptosis in cancer cells,perhaps because heightened activity of NOX or other pro-oxidant enzymes,or basally altered membrane lipid composition, is sufficient to promotedeath in the absence of these additional features. Regardless, theoxidative death pathways triggered in cancer cells and brain cells byblockade of cystine uptake both appear to access a core iron- andROS-dependent ferroptotic mechanism, accounting for the ability of Fer-1and CPX to attenuate death in both cases (FIG. 7E).

The specific role of iron in ferroptosis remains unclear. Ferroptosiscannot be explained by a simple increase in H₂O₂-dependent,iron-catalyzed ROS production (i.e. Fenton chemistry), as H₂O₂-induceddeath is distinct from RSL-induced ferroptosis (FIGS. 1 and 2). Rather,the results are most consistent with one or more iron-dependent enzymesfunctioning as part of the core, oxidative lethal mechanism. The voidcreated in the antioxidant defenses of the cell by the inhibition ofcystine uptake by erastin may be required to unleash the activity ofthese enzymes. Thus, for better or worse, the aberrantly elevated levelsof iron observed in some cancer cells (Pinnix et al., 2010) andpathological neuronal populations (Duce et al., 2010; Lei et al., 2012)may predispose to ferroptotic death in situations of cystine or cysteinelimitation.

Example 10 Fer-1 and its Analogs are Able to Inhibit Death in ErastinTreated Cells

The ability of various compounds disclosed herein to inhibit death inerastin (10 μM)-treated HT-1080 cells were tested. The results are shownin Table 1 below. Cell viability was assessed by Alamar Blue. EC₅₀values (nM) were computed from dose response curves.

TABLE 1 EC50 Values Compound EC50 (nM) SRS8-28 (Fer-1) 95 SRS15-15 Notactive SRS15-18 87.7 SRS15-23 23 SRS15-24 8.6 SRS15-25 54.3 SRS15-17171.4 SRS15-20_mono 21.7 SRS15-20_di 914 SRS15-21 23 SRS15-22 21.1SRS15-72 288 SRS16-41 16 SRS16-80 583 SRS16-86 367

Example 11 Metabolic Stability in Microsomes

Test compounds (0.5 μM) were incubated at 37° C. for up to 45 minutes in50 mM of potassium phosphate buffer (pH 7.4) containing mouse livermicrosomal protein (0.5 mg/mL) and an NADPH generating system (0.34mg/mL β-nicotinamide adenine dinucleotide phosphate (NADP), 1.56 mg/mLglucose-6-phosphate, 1.2 units/mL glucose-6-phosphate dehydrogenase). At0, 5, 15, 30, and 45 minute intervals, an aliquot was taken and quenchedwith acetonitrile (ACN) containing internal standard. No-cofactorcontrols at 45 minutes were prepared. Following completion of theexperiment, the samples were analyzed by LC-MS/MS.

The half-life (t_(1/2)) was calculated using the following equation:t _(1/2)=0.693/kWhere, k is the elimination rate constant of test compounds obtained byfitting the data to the equation:C=initial×exp(−k×t)Intrinsic clearance (Clint) is calculated as liver clearance from thehalf-life using the following equation:

${{CL}\mspace{14mu}{int}} = {\frac{rate}{\min^{- 1}} \times \frac{ml}{0.5\mspace{14mu}{mg}} \times \frac{52.5\mspace{14mu}{mg}}{g\mspace{14mu}{liver}}}$Where:

-   -   rate=k/min    -   0.5 mg protein/mL incubation    -   52.5 mg protein/g liver

The results are shown in Table 2 below. Ethoxycoumarin was used as acontrol.

TABLE 2 Microsomal Stability (Mouse, Compound Conc = 0.5 μM) IntrinsicClearance Elimination rate Half life (CLint) (mL/min/g Test compoundconstant (k) (t½) (min) liver) SRS16-80 0.0089 77.7 0.94 SRS16-86<0.0048 >90 <0.5* SRS15-72 0.0045 154.0 0.47 SRS 16-41 0.3149 2.0 35.9Ethoxycoumarin 0.2661 2.6 27.94 *Rate constant was negative (−0.0032mL/min/g liver) **: Half life more than 90 min will be reported as >90;and less than 1.4 will be reported as <1.4. ***: Rate constants formicrosomes less than 0.0048 or negative will be reported as <0.0048; andmore than 0.48 will be reported as >0.48.

Example 12 In Vitro Stability Study in Plasma

The assay was conducted by incubating 1 μM of test compounds with MousePlasma (Strain C57BL/4) for a series of incubation times (0, 15, 30, 60,120 minutes) at various temperatures (wet ice, room temperature, or 37°C.) with 5% CO₂ and shaking (130 rpm). At each time point, the sampleswere quenched with 200 μL of stop solution containing an internalstandard and kept on ice. The samples were analysed by LC-MS/MS. Theresults are shown in Tables 3 and 4 below.

TABLE 3 37° C. Compound Time points Sum of Peak Percent Name (minutes)Area Ratios** Remaining* SRS16-80 0 0.382 100.0% SRS16-80 15 0.091123.8% SRS16-80 30 0.0379 9.9% SRS16-80 60 0.017 4.5% SRS16-80 120 0.00952.5% SRS16-86 0 0.9018 100.0% SRS16-86 15 0.7414 82.2% SRS16-86 30 0.7886.5% SRS16-86 60 0.7568 83.9% SRS16-86 120 0.5758 63.9% *Percentremaining: Percentage of peak area at each time point compared to peakarea at 0 min. **Two peaks were seen for both compounds, which arebelieved to be the isomers. Hence, sum of the peaks were used forcalculation.

TABLE 4 Room Temperature Wet Ice Time Peak Peak points Area Percent AreaPercent Compound (min) Ratio Remaining* Ratio Remaining SRS 15-72 02.0737 100.0% 2.6066 100.0% SRS 15-72 15 1.1581 55.8% 2.0815 79.9% SRS15-72 30 0.5320 25.7% 1.6279 62.5% SRS 15-72 60 0.2502 12.1% 1.439755.2% SRS 15-72 120 0.0841 4.1% 1.2446 47.7% SRS 16-41 0 5.8556 100.0%6.9233 100.0% SRS 16-41 15 0.6709 11.5% 5.7631 83.2% SRS 16-41 30 0.08851.5% 4.7595 68.7% SRS 16-41 60 0.0206 0.4% 3.4547 49.9% SRS 16-41 1200.0159 0.3% 1.9564 28.3%As demonstrated by Examples 10-12, the Fer-1 analogs show improvedmicrosomal stability and solubility while maintaining good inhibitionpotency of ferroptosis.

Example 13 Fer-1, SRS11-92, and Analogs Thereof are Potent Inhibitors ofa Variety of Clinically Relevant Oxidative Cell Death Phenotypes

Fer-1 (disclosed in International Application Serial No.PCT/US/2013/035021, filed Apr. 2, 2013, which is incorporated byreference in its entirety herein) is a drug-like, easily synthesized,potent inhibitor of ferroptosis that acts via a reductive mechanism tospecifically prevent the loss of unsaturated membrane lipids, whoseoxidative destruction is presumably toxic to cells (see below). Fer-1 isan alkyl, diaminoaryl-type structure. The structurally relateddiarylamines and hindered dialkylamines are well-establishedantioxidants (also known as scavengers or reducing agents) used in thefood industry and in the materials industry (Huang, et al., 2005). Theseantioxidants retard autoxidation that is caused primarily by radicalchain reactions between oxygen and the substrates. There has been atremendous interest in investigating the effect of phenols,diphenylamines and hindered alkylamines as potential antioxidants inbiological and medicinal systems toward the search of novel therapeutics(Behl, et al., 1999). Most studies of amine antioxidants have beenfocused either on diarylamines or hindered dialkylamines. Here, a thirdsubfamily of antioxidants, the alkyl, diaminoarenes, represented byFer-1, is described.

Fer-1 attenuates lipid peroxidation and ferroptosis in cancer cellstreated with small molecules that block cystine import or glutathioneproduction, as well as cell death in rat organotypic hippocampal brainslices treated with toxic concentrations of glutamate, which also blockscystine import (Dixon, et al., 2012). Therefore, Fer-1 could beeffective at preventing iron-dependent oxidative stress and/or glutamatetoxicity in other contexts. Huntington's disease (HD), like otheraging-associated diseases, is reported to involve perturbation ofglutamate (Petr, et al., 2013, Ribeiro, et al., 2011, Anitha, et al.,2011, Miller, et al., 2010, Estrada Sanchez, et al., 2008, Zeron, etal., 2002, Cha, et al., 1998), glutathione (Mason, et al., 2013,Ribeiro, et al., 2012, Cruz-Aguado, et al., 2000) and iron (Chen, etal., 2013, Bartzokis, et al., 1999, Morrison, et al., 1994, Chen, etal., 1993) levels, the accumulation of lipid oxidation breakdownproducts (Johri, et al., 2012), and non-apoptotic neuronal cell death(Turmaine, et al., 2000). In fact, Fer-1 prevents cell death in ratcorticostriatal brain slices induced by the expression, via biolistictransformation, of a huntingtin (htt) exon 1 fragment with a pathogenicrepeat (73Q) (mN90Q73), along with yellow fluorescent protein (YFP) tomark transfected neurons (FIG. 15A). Slices were treated with DMSO(vehicle control), a positive control death inhibitor combination of theadenosine A2R receptor antagonist KW-6002 (KW, 50 μM) and the JNKinhibitor SP600125 (SP, 30 μM), or ferrostatins (Fer-1 or SRS11-92, aneffective analog, 1 nM to 1 μM). Four days later, the number of healthymedium spiny neurons (MSNs) was quantified, as previously described(Varma, et al., 2011, Hoffstrom, et al., 2010). A significant increasein the number of healthy MSNs was observed upon Fer-1 and SRS11-92treatment at 10 nM, 100 nM and 1 μM. Moreover, with 1 μM treatment ofFer-1, the number of healthy MSNs was statistically indistinguishablefrom both the YFP (no htt) control and the control inhibitor combination(KW+SP) (FIG. 15A).

Fer-1 was also found to be effective in an in vitro model ofperiventricular leukomalacia (PVL), a syndrome afflicting prematureinfants that is caused primarily by the death of developingoligodendrocytes (OLs) (Desilva, et al., 2008, Follett, et al., 2004,Dommergues, et al., 1998) (FIG. 15B). It has been suggested that OLdeath is iron-dependent (Dommergues, et al., 1998); in autopsy studies,elevated levels of lipid ROS biomarkers are observed, potentiallysuggesting the involvement of ferroptosis (Desilva, et al., 2008,Follett, et al., 2004, Folkerth, et al., 2006, Haynes, et al., 2005,Haynes, et al., 2003). To trigger death, cultured OLs were exposed tocystine-free conditions, which ultimately deplete glutathione. Fer-1,SRS11-92 and Fer-1 analogs fully protected OLs from cystine deprivationwhen tested at 100 nM (FIG. 15B).

In addition, the efficacy of Fer-1, SRS11-92, and selected analogs wastested in a model of iron-induced cell death in freshly isolated mousekidney proximal tubules that simulates major elements ofrhabdomyolysis-induced acute kidney injury (FIG. 15C). Cell death inthis model is known to involve oxidative lipid damage (Sogabe, et al.,1996, Park, et al., 2011); it was therefore believed that Fer-1 might beprotective. 10 μM each of hydroxyquinoline and ferrous ammonium sulfate(HQ+Fe) were used to induce cell death, which was quantified bymonitoring LDH release after 60 min. In this model, Fer-1, SRS11-92 andanalogs prevented lethality (FIG. 15C). Together, the results of theseassays demonstrate that Fer-1, SRS11-92 and analogs are potentinhibitors of a variety of clinically relevant oxidative cell deathphenotypes. By implication, cell death in these models may involve, atleast in part, the induction of lipid peroxidation and perhapsferroptosis.

Example 14 Fer-1 is not a General Antioxidant

Given that ROS also play important homeostatic roles, an importantconsideration in the design of antioxidants is specificity: that theyonly inhibit pathogenic ROS. Fer-1 was found to be able to preventspecific forms of ROS accumulation. In HT-1080 cancer cells, Fer-1 didnot prevent the accumulation of mitochondrial superoxide in response tothe electron transport chain inhibitor rotenone (detected using thefluorescent probe MitoSOX) (FIG. 16A), mitochondrial cardiolipinoxidation in response to the apoptosis inducing agent staurosporine(detected using the fluorescent probe 10-N-nonylacridine orange) (FIG.16B), or lysosomal membrane destruction in response to H₂O₂ (detected byfollowing acridine orange re-localization) (FIG. 16C), consistent withthe inability of Fer-1 to prevent cell death involving mitochondrial ROSoverproduction, apoptosis or lysosome rupture in these cells. Theseresults revealed that Fer-1 is not a general antioxidant; the effect ofFer-1 is restricted to certain ROS, membranes and/or membrane lipids.

Example 15 Ferrostatin Structure Activity Relationships and ImprovedPharmacokinetic/Pharmacodynamic Properties

Solvents, inorganic salts, and organic reagents were purchased fromcommercial sources such as Sigma and Fisher and used without furtherpurification unless otherwise mentioned. Erastin was dissolved in DMSOto a final concentration of 73.1 mM and stored in aliquots at −20° C.

Chromatography: Merck pre-coated 0.25 mm silica plates containing a 254nm fluorescence indicator were used for analytical thin-layerchromatography. Flash chromatography was performed on 230-400 meshsilica (SiliaFlash® P60) from Silicycle.

Spectroscopy: ¹H, ¹³C and ¹⁹F NMR spectra were obtained on a Bruker DPX400 MHz spectrometer. HRMS spectra were taken on double focusing sectortype mass spectrometer HX-110A. Maker JEOL Ltd. Tokyo Japan (resolutionof 10,000 and 10 KV accel. Volt. Ionization method; FAB (Fast AtomBombardment) used Xe 3Kv energy. Used Matrix, NBA (m-Nitro benzylalcohol).

General Procedure A (ArS_(N)2 Reaction) (Beaulieu et al., 2003)

To the ethyl 4-chloro-3-nitrobenzoate (1 equiv., 200 mg, 0.871 mmol) indry DMSO (2 mL) was added K₂CO₃ (2 equiv., 240.8 mg, 1.742 mmol) andvarious amines (1.2 equiv., 119.5 μL, 1.045 mmol). The mixture wasstirred for 17 hours at 60° C. The solution was poured in water, and theorganic layer was extracted three times with ethyl acetate. After dryingwith anhydrous magnesium sulfate, the solvents were removed undervacuum. The residue was purified by flash-column chromatography onsilica gel to provide the desired ethyl4-(substituted-amino)-3-nitrobenzoate derivatives.

General Procedure B (Hydrogenolysis)

The ethyl 4-(substituted-amino)-3-nitrobenzoates (130 mg, 0.445 mmol)were dissolved in MeOH (10 mL) and hydrogenated (H₂ gas) over 10%Pd(OH)₂ on charcoal (90 mg) for 17 hours at room temperature. Thesolution was filtered through a pad of celite, and volatiles wereremoved under vacuum. The residue was purified by flash-columnchromatography on silica gel to provide the desired Ferrostatin-1derivatives.

General Procedure C (Reductive Amination Reaction) (Abdel-Magid et al.,1996)

A representative example is the reductive amination of Fer-1 withbenzaldehyde.

Method I: the ethyl 3-amino-4-(cyclohexylamino)benzoate (Fer-1) (100 mg,0.382 mmol, 1 equiv) and benzaldehyde (39 μL, 0.382 mmol, 1 equiv) wereheated in DCE for 1 hour at 80° C. in the presence of molecular sieve (4Å), and then the mixture was cooled down to room temperature beforeaddition of the NaBH(OAc)₃ in small portions over 3 hours. The reactionmixture was stirred at room temperature under a nitrogen atmosphere for17 hours. The reaction mixture was quenched with aqueous saturatedNaHCO₃, and the product was extracted with EtOAc. The EtOAc extract wasdried (MgSO₄), and the solvent was evaporated. The residue was purifiedby flash-column chromatography on silica gel to provide the desiredethyl 3-(benzylamino)-4-(cyclohexylamino)benzoate.

Method II: To the ethyl 3-amino-4-(cyclohexylamino)benzoate (Fer-1) (100mg, 0.382 mmol, 1 equiv) and benzaldehyde (39 μL, 0.382 mmol, 1 equiv)in DCE was added NaBH(OAc)₃ (129.5 mg, 0.611 mmol, 1.6 equiv). Thereaction mixture was treated in the same way as in method I.

General Procedure D (Alkylation Reaction)

A representative example is the methylation of the SRS8-70 (Table 1,entry 18) using methyl iodide. To the ethyl3-amino-4-(cyclooctylamino)benzoate (SRS8-70; 58 mg, 0.199 mmol) in DMF(1 mL), MeI (28 μL, 0.398 mmol) and K₂CO₃ (82 mg, 0.508 mmol) wereadded. The mixture was stirred at 40° C. for 6 hours then poured inwater. The organic layer was extracted with EtOAc then dried underMgSO₄, and the solvent was evaporated. The residue was purified byflash-column chromatography on silica gel to provide the desired ethyl4-(cyclooctylamino)-3-(dimethylamino)benzoate (SRS9-01).

General Procedure E (Addition of the Fer-1 to an Acylchloride, Alkyl-,Benzyl-Chloroformates)

A representative example is the addition of aniline of the Fer-1 to thebenzylchloroformate. To the ethyl 3-amino-4-(cyclohexylamino)benzoate(Fer-1; SRS8-28; 22 mg, 0.084 mmol) in THF (1 mL), benzylchloroformate(24 μL, 0.168 mmol) and DIPEA (44 μL, 0.252 mmol) were added at 0° C.The mixture was stirred at room temperature for 17 hours then poured inwater. The organic layer was extracted with EtOAc then dried underMgSO₄, and the solvent was evaporated. The residue was purified byflash-column chromatography on silica gel to provide the desired ethyl3-(benzyloxycarbonylamino)-4-(cyclohexylamino)benzoate (SRS11-89, SITable 5, entry 2).

A general route to ferrostatin analogs using a three-step synthesisgenerated 67 analogs. An SNAr reaction between the commerciallyavailable ethyl 4-chloro-3-nitrobenzoate and cyclohexylamine, followedby catalytic hydrogenolysis of the nitro group, provided the desiredferrostatin derivatives. The anilines of the latter were reacted throughreductive amination with arylaldehydes in the presence of sodiumtriacetoxyborohydride or through straightforward alkylation witharylalkylhalides in the presence of Hunig's base (Scheme 5). Examinationof the Fer-1 structure suggests a possible release of two protons andtwo electrons, resulting in the formation of a redox-stable compound B(Scheme 6). This mechanism could form the basis for the antioxidantscavenging ability of Fer-1; the imine analog SRS16-86 is likely reducedin cells if it acts through this mechanism.

TABLE 2 SI, Synthetic scheme of Ferrostatin-1 analogs with varioushydrophobic moieties. See, e.g., Dixon et al., Cell 2012, 149,1060-1072.

R₁ R₂ Name (Yield) EC50 (nM) Log P  1

H Fer-1 (SRS8-28)¹ (85%)  95 3.2  2

H SRS8-72¹ (74%) 880 1.9  3

H SRS8-71¹ (80%) 265 2.3  4

H SRS8-73¹ (85%) 120 2.7  5

H SRS8-42¹ (64%) 200 2.9  6

H SRS8-48¹ (89%) (80% as mixture of isomers)  90 3.5  7

H SRS13-10_F1_ single isomer SRS13-10_F2_ single isomer SRS13-10¹_F1+F2_mixture of isomers  44    13    62 4.7  8

H SRS8-90¹ (86%) 130 3.6  9

H SRS8-70¹ (89%)  80 4.1 10

H SRS8-94¹ (90%)  80 4.3 11

H SRS9-06¹ (80%)  70 5.8

Synthesis of ethyl 3-amino-4-(phenylamino)benzoate (SRS8-42. SI, Table2)

¹H NMR (400 MHz, CDCl₃) δ 7.52 (d, J=7.9 Hz, 2H), 7.30 (t, J=7.9 Hz,2H), 7.19 (d, J=8.1 Hz, 1H), 6.97 (d, J=8.3 Hz, 3H), 5.74 (s, NH), 4.37(q, J=7.1 Hz, 2H), 3.70 (s, NH₂), 1.41 (t, J=7.1 Hz, 3H); ¹³C NMR (126MHz, CDCl₃) δ 166.8, 142.9, 137.9, 135.6, 129.4, 124.6, 121.8, 121.3,118.4, 117.9, 60.7, 14.4.

Synthesis of ethyl 3-amino-4-(4-methylcyclohexylamino)benzoate (SRS8-48.SI, Table 2)

¹H NMR (CDCl₃, 400 MHz) δ 7.58 (d, J=8.3 Hz, 1H), 7.41 (s, 1H), 6.59 (d,J=8.4 Hz, 1H), 4.30 (d, J=7.1 Hz, 2H), 3.26 (s, 1H), 2.13 (d, J=11.2 Hz,2H), 1.78 (d, J=12.3 Hz, 2H), 1.35 (t, J=7.0 Hz, 4H), 1.23-1.07 (m, 3H),0.93 (d, J=6.4 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 166.98, 141.15,132.14, 124.13, 119.19, 118.69, 110.50, 60.25, 52.21, 33.91, 33.11,32.23, 22.20, 14.46; HRMS (FAB) calculated for C₁₆H₂₄N₂O₂: 276.37;found: 276.2.

Synthesis of single isomer of ethyl3-amino-4-(4-tert-butylcyclohexyl-amino)benzoate (SRS13-10 F1. SI, Table2)

¹H NMR (CDCl₃, 400 MHz) δ 7.49 (d, J=8.4 Hz, 1H), 7.43 (s, 1H) 0.6.61(d, J=8.4 Hz, 1H), 4.32 (q, J=7.1 Hz, 2H), 3.86 (b, 1H), 3.25 (b,1H+NH₂), 2.26-2.13 (m, 2H), 1.95-1.81 (m, 2H), 1.43-1.33 (m, 3H),1.22-1.12 (m, 4H), 1.10-1.04 (m, 1H), 0.89 (s, 9H); ¹³C NMR (100 MHz,CDCl₃) δ 167.0, 142.3, 131.67, 124.3, 118.6, 118.4, 109.52, 60.2, 52.0,47.7, 33.82, 32.4, 27.6, 26.2, 14.5; HRMS (FAB) calculated forC₁₉H₃₀N₂O₂: 318.45; found: 318.24.

Synthesis of single isomer of ethyl3-amino-4-(4-tert-butylcyclohexyl-amino)benzoate (SRS13-10 F2. SI, Table2)

¹H NMR (CDCl₃, 400 MHz) δ 7.66-7.56 (m, 1H), 7.44 (s, 1H). 6.65-6.56 (m,1H), 4.39-4.26 (m, 2H), 3.75 (s, 1H), 3.22 (B, NH₂), 2.10-2.96 (m, 2H),1.70-1.52 (m, 4H), 1.43-1.34 (m, 3H), 1.30-1.20 (m, 2H), 1.13-1.05 (m,1H), 0.88 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 167.1, 142.4, 131.9,124.5, 118.7, 118.4, 109.6, 60.1, 47.8, 46.5, 32.5, 30.3, 27.4, 21.6,14.45; HRMS (FAB) calculated for C₁₉H₃₀N₂O₂: 318.45; found: 318.24.

Synthesis of ethyl 3-amino-4-(octylamino)benzoate (SRS8-94. SI, Table 2)

¹H NMR (CDCl₃, 400 MHz) δ 7.61 (d, J=8.3 Hz, 1H), 7.43 (d, J=2.0 Hz,1H), 6.60 (d, J=8.3 Hz, 1H), 4.33 (q, J=7.1 Hz, 2H), 3.17 (t, J=7.1 Hz,2H), 1.69 (q, J=7.3 Hz, 2H), 1.52-1.23 (m, 13H), 0.91 (t, J=6.6 Hz, 3H);¹³C NMR (100 MHz, CDCl₃) δ 167.1, 143.2, 131.9, 124.3, 118.9, 118.1,109.2, 60.2, 43.7, 31.8, 29.4, 29.3, 27.2, 22.7, 14.5, 14.1; HRMS (FAB)calculated for C₁₇H₂₈N₂O₂: 292.42. found: 292.22.

Synthesis of ethyl 3-amino-4-(cyclododecylamino)benzoate (SRS9-06. SI,Table 2)

¹H NMR (400 MHz, CDCl₃) δ 7.62 (d, J=8.4 Hz, 1H), 7.43 (s, 1H), 6.60 (d,J=9.6 Hz, 1H), 4.32 (d, J=9.6 Hz, 2H), 3.19 (s, 1H), 1.43 (d, J=26.7 Hz,25H), 1.28 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 167.1, 142.6, 131.7,124.5, 118.6, 118.16, 109.2, 60.2, 49.2, 29.6, 24.33, 24.0 23.3, 23.2,21.22, 14.5; HRMS (FAB) calculated for O₂₁H₃₄N₂O₂: 346.51; found:346.29.

TABLE 3 SI, EC50 and Log P of Ferrostatin-1 analogs with various cyclicamines bearing heteroatoms.

Name EC50 Entry R₁ R₂ (Yield) (nM) Log P 1

H SRS8-41 (85%) 1450 1.8 2

H SRS8-54 (50%) 2500 1.9 3

H SRS8-47 (65%)  710 2.8 4

H SRS8-81 (86%)  350 1.5 5

H SRS8-46 (81%0  515 3.6 6

H SRS8-80 (81%)  380 1.9 7

H SRS8-53 (70%) 3600 1.7 8

H SRS8-52 (87%)  650 3.8

Synthesis of ethyl4-(2-amino-4-(ethoxycarbonyl)phenylamino)piperidine-1-carboxylate(SRS8-54. SI, Table 3)

¹H NMR (400 MHz, CDCl₃) δ 7.51 (d, J=8.0 Hz, 1H), 7.36 (s, 1H), 6.55 (d,J=7.8 Hz, 1H), 4.28-4.21 (m, NH2), 4.12-4.00 (m, 4H), 3.47 (s, 1H), 2.96(s, 2H), 1.98 (s, 2H), 1.44-1.11 (m, 10H); ¹³C NMR (100 MHz, CDCl₃) δ167.0, 155.5, 141.1, 132.5, 123.8, 119.0, 118.3, 109.5, 61.4, 60.2,49.5, 42.6, 32.0, 14.7, 14.4; HRMS (FAB) calculated for C₁₇H₂₅N₃O₄:335.40; found: 335.18.

Synthesis of tert-butyl4-(2-amino-4-(ethoxycarbonyl)phenylamino)-piperidine-1-carboxylate(SRS8-47. SI, Table 3)

¹H NMR (400 MHz, CDCl₃) δ 7.57 (d, J=10.1 Hz, 1H), 7.42 (s, 1H), 6.60(d, J=8.4 Hz, 1H), 4.31 (q, J=7.1 Hz, 2H), 3.26 (s, 2H), 2.97 (t, J=11.7Hz, 2H), 2.05 (d, J=12.6 Hz, 2H), 1.41 (d, J=45.5 Hz, 16H); ¹³C NMR (100MHz, CDCl₃) δ 167.0, 154.7, 141.3, 132.3, 124.0, 119.0, 118.5, 109.6,77.0, 60.2, 53.5, 49.6, 32.1, 28.4, 14.4; HRMS (FAB) calculated forC₁₉H₂₉N₃O₄: 363.45; found: 363.21.

Synthesis of ethyl 3-amino-4-(cyclohexylamino)benzoate (SRS8-81. SI,Table 3)

¹H NMR (400 MHz, CDCl₃) δ 7.55 (d, J=8.2 Hz, 1H), 7.41 (s, 1H), 6.57 (d,J=8.2 Hz, 1H), 4.30 (d, J=6.8 Hz, 2H), 3.49 (s, 1H+NH₂), 3.24 (b, NH),2.83 (s, 2H), 2.11 (s, 2H), 1.55 (d, J=9.2 Hz, 2H), 1.35 (s, 3H), 1.26(s, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 167.0, 141.4, 132.1, 124.1, 119.0,118.6, 109.6, 60.3, 49.5, 44.9, 32.9, 29.7, 14.5; LC/MS (APCI+, M+1)264.67

Synthesis of ethyl3-amino-4-(4-(tert-butoxycarbonylamino)cyclohexyl-amino)benzoate(SRS8-46. SI, Table 3)

¹H NMR (400 MHz, CDCl₃) δ 7.55 (d, J=8.1 Hz, 1H), 7.39 (s, 1H),6.56-6.51 (m, 1H), 4.55 (s, 1H), 4.29 (d, J=9.9 Hz, 2H), 3.45 (s, 2H),3.25 (s, 1H), 2.09 (d, J=26.0 Hz, 4H), 1.44 (s, 9H), 1.29 (d, J=30.4 Hz,7H); ¹³C NMR (100 MHz, CDCl₃) δ 167.0, 155.3, 141.8, 132.0, 124.1,118.7, 118.5, 109.4, 79.2, 60.2, 50.9, 49.2, 32.0, 31.8, 28.4, 14.5;HRMS (FAB) calculated for C₂₀H₃₁N₃O₄: 377.48. found: 377.23.

Synthesis of ethyl 3-amino-4-(4-aminocyclohexylamino)benzoate (SRS8-80.SI, Table 3)

¹H NMR (400 MHz, MeOD) 7.45 (d, J=8.3 Hz, 1H), 7.38 (s, 1H), 6.62 (d,J=8.4 Hz, 1H), 4.27 (d, J=7.1 Hz, 2H), 3.41 (s, 1H), 2.18 (s, 2H), 2.04(s, 2H), 1.37 (t, J=7.1 Hz, 4H), 1.31 (s, 3H); ¹³C NMR (100 MHz, MeOD) δ167.8, 141.0, 132.7, 122.8, 117.5, 116.79, 108.8, 59.9, 50.4, 49.7,31.78, 30.82, 13.33; HRMS (FAB) calculated for C₁₅H₂₃N₃O₂: 277.36;found: 277.18.

Synthesis of ethyl 3-amino-4-(4,4-difluorocyclohexylamino)benzoate(SRS8-52. SI, Table 3)

¹H NMR (400 MHz, CDCl₃) δ 7.63 (s, 1H), 7.46 (s, 1H), 6.63 (s, 1H), 4.37(s, 2H), 3.95 (s, 1H) 3.28 (s, 1H), 2.16 (s, 4H), 1.96 (d, J=13.7 Hz,2H), 1.68 (s, 2H), 1.37 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 166.8,141.5, 132.1, 124.3, 119.6, 119.0, 109.9, 60.3, 49.2, 31.9, 30.9, 28.7,14.4; ¹⁹F (CDCl₃) −94.9, −98.5; HRMS (FAB) calculated for C₁₅H₂₀F₂N₂O₂:298.33; found: 298.15.

Preparation of the Nitrobenzene Bearing Various Esters (SI, Table 4,Entries 2-7) and Amide (SI, Table 4, Entry 8)

A representative example is the coupling reaction between the4-chloro-3-nitrobenzoic acid and the 4-(2-hydroxyethyl)morpholine (SI,Scheme 1, 1c). To the 4-chloro-3-nitrobenzoic acid (500 mg, 2.487 mmol)in DCM, under nitrogen, was added the 4-(2-hydroxyethyl)morpholine (332μL, 2.736 mmol) and DMAP (91 mg, 0.467 mmol). The mixture was cooled to0° C. before the addition of the N,N′ Dicyclohexylcarbodiimide (DCC)(615 mg, 2.985 mmol). Then the reaction mixture was left at roomtemperature for 17 h. The precipitate was filtered over celite and theorganic solvent was evaporated. The residue was purified by flash-columnchromatography on silica gel to provide the desired 2-morpholinoethyl4-chloro-3-nitrobenzoate (SI, Scheme 1, 1c).

Synthesis of N1-cyclohexylbenzene-1,2-diamine (SRS8-75. SI, Table 4)

¹H NMR (CDCl₃, 400 MHz) δ 6.91-6.81 (m, 1H), 6.74 (dt, J=14.4, 6.6 Hz,3H), 3.30 (s, 1H), 2.12 (d, J=10.5 Hz, 2H), 1.83 (d, J=12.7 Hz, 2H),1.72 (d, J=9.0 Hz, 1H), 1.53-1.37 (m, 2H), 1.36-1.19 (m, 3H); ¹³C NMR(100 MHz, CDCl₃) δ 136.9, 134.5, 120.7, 118.3, 117.0, 112.8, 51.9, 33.8,26.2, 25.2; HRMS (FAB) calculated for C₁₂H₁₈N₂: 190.28; found 190.15.

Synthesis of methyl 3-amino-4-(cyclohexylamino)benzoate (SRS8-61. SI,Table 4)

¹H NMR (CDCl₃, 400 MHz) δ 6.64-6.57 (m, 1H), 7.42 (s, 1H), 6.64-6.57 (m,1H), 3.35 (s, 1H), 2.09 (d, J=11.4 Hz, 2H), 1.80 (d, J=12.6 Hz, 2H),1.69 (dt, J=12.9, 4.2 Hz, 1H), 1.42 (d, J=11.5 Hz, 2H), 1.31-1.20 (m,3H); ¹³C NMR (100 MHz, CDCl₃) δ 167.5, 142.2, 131.7, 124.4, 118.6,118.0, 109.5, 51.6, 51.3, 33.3, 25.8, 24.9; HRMS (FAB) calculated forC₁₄H₂₀N₂O₂: 248.32; found: 248.15.

Synthesis of decyl 3-amino-4-(cyclohexylamino)benzoate (SRS12-29. SI,Table 4)

¹H NMR (CDCl₃, 400 MHz) δ 7.59 (d, J=8.4 Hz, 1H), 7.42 (s, 1H), 6.61 (d,J=8.4 Hz, 1H), 4.27-4.24 (m, 2H), 3.35 (b, 1H), 2.10-2.07 (m, 2H),2.78-1.72 (m, 4H), 1.43-1.29 (m, 22H), 0.90-0.88 (m, 3H); ¹³C NMR (100MHz, CDCl₃) δ 167.0, 141.2, 132.2, 124.2, 119.2, 118.7, 110.4, 64.5,51.8, 33.1, 31.9, 29.55, 29.3, 28.9, 26.1, 25.8, 24.9, 22.7, 14.1; HRMS(FAB) calculated for C₂₃H₃₈N₂O₂: 374.56; found: 374.47.

Synthesis of 2-morpholinoethyl 3-amino-4-(cyclohexylamino)benzoate(SRS12-47. SI, Table 4)

¹H NMR (CDCl₃, 400 MHz) δ 7.56 (d, J=5.0 Hz, 1H), 7.39 (d, J=2.0 Hz,1H), 6.59 (d, J=8.4 Hz, 1H), 4.40 (t, J=6.0 Hz, 2H), 3.90 (b, NH),3.73-3.71 (m, 4H), 3.33 (b, 1H), 3.19 (b, NH₂), 2.76 (t, J=6.0 Hz, 2H),2.59-2.56 (m, 4H), 2.09-2.04 (m, 2H), 1.82-1.17 (m, 8H); 13C NMR (100MHz, CDCl₃) δ 166.8, 142.3, 131.8, 124.5, 118.6, 117.9, 109.4, 67.0,61.8, 57.3, 53.9, 51.3, 49.1, 34.0, 33.3, 25.8, 24.9; HRMS (FAB)calculated for C₁₉H₂₉N₃O₃: 347.45; found: 348.31.

Synthesis of 2-(2-(tert-butoxycarbonylamino)ethoxy)ethyl3-amino-4-(cyclohexylamino)benzoate (SRS8-37. SI, Table 4)

¹H NMR (400 MHz, CDCl₃) δ 7.58 (d, J=10.2 Hz, 1H), 7.42 (s, 1H), 6.59(d, J=8.4 Hz, 1H), 4.41 (s, 2H), 3.77 (s, 2H), 3.58 (d, J=4.7 Hz, 2H),3.33 (s, 3H), 2.07 (d, J=11.6 Hz, 2H), 1.78 (d, J=12.6 Hz, 2H),1.73-1.64 (m, 1H), 1.44 (s, 12H), 1.24 (s, 3H); ¹³C NMR (100 MHz, CDCl₃)δ 166.9, 156.0, 142.4, 131.7, 124.6, 118.7, 117.8, 109.5, 79.3, 70.2,69.2, 63.3, 51.3, 40.4, 33.3, 28.4, 25.9, 24.9; LC/MS (APCI+, M+1)421.69

Synthesis of 2-(2-aminoethoxy)ethyl 3-amino-4-(cyclohexylamino)benzoate(SRS8-43. SI, Table 4)

¹H NMR (400 MHz, CDCl₃) δ 7.46 (d, J=8.0 Hz, 1H), 7.39 (5, 1H), 6.70 (d,J=8.0 Hz, 1H), 4.48-4.41 (m, 2H), 3.95 (5, 4H), 3.84-3.80 (m, 2H),3.75-3.68 (m, 2H), 3.28 (5, 1H), 3.10-3.00 (m, 2H), 2.07 (d, J=11.1 Hz,2H), 1.79 (d, J=9.5 Hz, 2H), 1.69 (d, J=8.6 Hz, 1H), 1.41 (d, J=11.7 Hz,2H), 1.29-1.18 (m, 3H); LC/MS (APCI+, M+1) 321.69

Synthesis of tert-butyl 3-amino-4-(cyclooctylamino)benzoate (SRS8-87.SI, Table 4)

¹H NMR (CDCl₃, 400 MHz) δ 7.53 (dd, J=8.3, 1.9 Hz, 1H), 7.35 (d, J=1.9Hz, 1H), 6.51 (d, J=8.4 Hz, 1H), 3.20 (s, 1H), 1.91 (s, 2H), 1.75 (d,J=7.7 Hz, 2H), 1.56 (s, 19H); ¹³C NMR (100 MHz, CDCl₃) δ 166.4, 141.6,132.0, 124.0, 120.0, 118.3, 109.7, 79.7, 52.3, 32.7, 28.4, 27.0, 26.0,24.1; HRMS (FAB) calculated for C₁₉H₃₀N₂O₂: 318.45; found: 318.23.

Synthesis of 3-amino-4-(cyclooctylamino)-N-ethylbenzamide (SRS9-11. SI,Table 4)

¹H NMR (400 MHz, CDCl₃) δ 7.25 (d, J=2.1 Hz, 1H), 7.20 (dd, J=8.3, 2.1Hz, 1H), 6.52 (d, J=8.3 Hz, 1H), 5.93 (s, 1H), 3.54 (s, 1H), 3.48-3.44(m, 2H), 1.90 (s, 2H), 1.76 (d, J=8.1 Hz, 1H), 1.60 (s, 11H), 1.23 (t,J=7.3 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 167.5, 140.3, 132.9, 123.2,119.9, 116.4, 110.2, 52.5, 34.7, 32.8, 27.0, 26.0, 24.1, 15.1; LC/MS(APCI+, M+1) 389.83

TABLE 4 SI, Synthetic scheme of Ferrostatin-1 analogs with various R₃substitutions.

Entry R₁ R₂ R₃ Name (Yield) EC50 (nM) Log P 1

H H SRS8-75 (86%) 330 2.9 2

H

SRS8-61 (90%) 150 2.8 3

H

SRS12-29 (92%)  75 5.9 4

H

SRS12-47 (81%) 660 2.3 5

H

SRS8-37 (98%) 160 3.2 6

H

SRS8-43 (89%) 420 1.6 7

H

SRS8-87 (87%)  90 4.9 8

H

SRS9-11 (66%) 950 3.4

TABLE 5 SI, EC50 and Log P of Ferrostatin-1 analogs.

Entry/Name (Yield) R1 R₂ R₃ R₄ EC50 (nM) Log P  1 Ferrostatin-1(SRS8-28; 85%)

H H H 88 3.2  2 SRS11-89 (88%)¹

H H

>10,000 5.2  3 SRS11-97 (86%)¹

H H

>10,000 3.4  4 SRS11-98 (70%)¹

H H

>10,000 3.1  5 SRS8-91 (95%)²

H H

>10,000 5.5  6 SRS8-70 (92%)

H H H 69 4.1  7 SRS9-14 (70%)³

H H CH₃ 70 4.4  8 SRS9-01 (92%)⁴

H CH₃ CH₃ 3000 4.6  9 SRS8-94 (95%) C₈H₁₇ H H H 80 4.4 10 SRS12-12 (89%)C₈H₁₇ C₈H₁₇ H H 15267 7.8 Conditions. ¹Addition of Fer-1 toacylchloride, alkyl- or benzyl-chloroformates (1 equiv.), DIPEA, DCM,R.T., 17 h. ²(Boc)₂O (1 equiv.), DMAP (cat.), THF, R.T., 17 h. ³usingreductive amination conditions (H₂CO (1 equiv.), NaBH(OAc)₃ (1.2-1.6equiv.), DCE, R.T., 17 h). ⁴MeI (2.2 equiv), K₂CO₃, DMF, 40° C., 17 h.

Synthesis of ethyl3-(benzyloxycarbonylamino)-4-(cyclohexylamino)-benzoate (SRS11-89. SI,Table 5)

¹H NMR (400 MHz, CDCl₃) δ 7.83 (d, J=10.3 Hz, 2H), 7.38 (s, 5H), 6.66(d, J=8.5 Hz, 1H), 4.34-4.11 (m, 4H), 3.32 (s, 1H), 1.99 (s, 2H), 1.74(s, 2H), 1.64 (s, 1H), 1.36 (t, J=7.1 Hz, 5H), 1.23-1.11 (m, 3H); ¹³CNMR (100 MHz, CDCl₃) δ 166.4, 154.6, 146.7, 136.0, 130.2, 128.6, 128.4,121.0, 117.9, 110.6, 67.5, 60.3, 51.3, 33.0, 25.7, 24.8, 14.5; HRMS(FAB) calculated for C₂₃H₂₈N₂O₄: 396.48; found: 396.20.

Synthesis of ethyl 4-(cyclohexylamino)-3-(methoxycarbonylamino)benzoate(SRS11-97. SI, Table 5)

¹H NMR (400 MHz, CDCl₃) δ 7.85 (s, 2H), 6.67 (d, J=9.0 Hz, 1H), 6.04 (s,1H), 4.32 (q, J=7.1 Hz, 2H), 3.78 (s, 2H), 3.36 (s, 1H), 2.06 (d, J=12.4Hz, 2H), 1.79 (d, J=13.4 Hz, 2H), 1.68 (d, J=12.6 Hz, 1H), 1.37 (t,J=7.1 Hz, 5H), 1.29-1.18 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 166.4,155.9, 146.7, 130.2, 129.1, 121.1, 117.9, 110.5, 60.3, 52.8, 51.3, 33.1,25.7, 24.8, 14.4; HRMS (FAB) calculated for C₁₇H₂₄N₂O₄: 320.38; found:320.17.

Synthesis of ethyl 4-(cyclohexylamino)-3-ethanamidobenzoate (SRS11-98.SI, Table 5)

¹H NMR (400 MHz, CDCl₃) δ 7.82 (d, J=8.6 Hz, 1H), 7.75 (s, 1H), 6.68 (d,J=8.6 Hz, 1H), 4.57-4.46 (m, 1H), 4.33 (d, J=9.8 Hz, 2H), 3.35 (s, 1H),2.22 (s, 3H), 2.04 (d, J=12.1 Hz, 2H), 1.88 (s, 1H), 1.79 (s, 3H), 1.38(s, 4H), 1.24 (d, J=8.6 Hz, 3H); HRMS (FAB) calculated for C₁₇H₂₄N₂O₃:304.38; found: 304.18.

Synthesis of ethyl3-(tert-butoxycarbonylamino)-4-(cyclooctylamino)-benzoate (SRS8-91. SI,Table 5)

¹H NMR (400 MHz, CDCl₃) δ 8.54 (d, J=1.5 Hz, 1H), 7.93 (dd, J=8.4, 1.7Hz, 1H), 7.08 (d, J=8.4 Hz, 1H), 4.44-4.35 (m, 2H), 2.23 (q, J=10.7 Hz,2H), 1.86 (d, J=9.0 Hz, 4H), 1.70 (s, 8H), 1.64-1.56 (m, 6H), 1.42-1.38(m, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 166.4, 150.5, 148.6, 132.0, 125.9,123.9, 115.8, 108.6, 85.0, 61.0, 31.9, 28.1, 27.8, 27.0, 26.2, 26.0,25.3, 14.4; HRMS (FAB) calculated for C22H34N2O4: 390.52;

Synthesis of ethyl 4-(cyclooctylamino)-3-(methylamino)benzoate (SRS9-14.SI, Table 5)

¹H NMR (400 MHz, CDCl₃) δ 7.60 (d, J=8.0 Hz, 1H), 7.37 (s, 1H), 6.54 (d,J=8.3 Hz, 1H), 4.34 (q, J=7.1 Hz, 2H), 3.56 (d, J=9.7 Hz, 1H), 2.90 (s,3H), 1.94-1.86 (m, 2H), 1.80 1.72 (m, 2H), 1.67-1.53 (m, 10H), 1.38 (t,J=7.1 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 167.5, 141.4, 136.3, 123.0,119.0, 112.6, 109.2, 60.2, 52.3, 32.8, 31.4, 27.0, 26.0, 24.1, 14.5;HRMS (FAB) calculated for C₁₈H₂₈N₂O₂: 304.43; found: 304.21.

Synthesis of ethyl 4-(cyclooctylamino)-3-(dimethylamino)benzoate(SRS9-01. SI, Table 5)

¹H NMR (100 MHz, CDCl₃) δ 7.78-7.70 (m, 2H), 6.50 (d, J=8.5 Hz, 1H),4.33 (q, J=7.1 Hz, 2H), 3.58 (s, 1H), 2.64 (s, 6H), 1.92 (s, 2H), 1.63(d, J=23.7 Hz, 12H), 1.38 (t, J=7.1 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ167.2, 146.0, 139.3, 128.0, 121.0, 117.0, 109.0, 60.1, 52.0, 44.0, 32.5,27.2, 25.8, 24.0, 14.5; HRMS (FAB) calculated for C₁₉H₃₀N₂O₂: 318.45;found 318.23.

Synthesis of ethyl 3-amino-4-(dioctylamino)benzoate (SRS12-12. SI, Table5)

¹H NMR (400 MHz, CDCl₃) δ 7.41 (s, 2H), 7.03 (d, J=8.7 Hz, 1H), 4.34 (d,J=7.1 Hz, 2H), 2.95 (s, 3H), 1.38 (d, J=7.1 Hz, 6H), 1.24 (s, 22H), 0.87(s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 166.9, 142.8, 142.3, 126.0, 121.8,119.7, 116.1, 60.6, 52.8, 31.8, 29.4, 29.3, 27.2, 22.6, 14.4, 14.1;LC/MS (APCI+, M−1) 403.96

TABLE 6 SI, EC50 and Log P of Ferrostatin-1 analogs.

Name EC50 Entry R1 (Yield) (nM) Log P  1

SRS11-92 (98%)   6 5.3  2

SRS12-58 (90%)  41 5.9  3

SRS12-49 (91%) 371 5.9  4

SRS12-35 (89%)  44 6.1  5

SRS12-57 (85%) 100 5.5  6

SRS12-33 (85%)  50 5.4  7

SRS12-48 (86%)  46 6.2  8

SRS12-50 (92%) 100 4.9  9

SRS12-71 (94%) 126 5.2 10

SRS12-36 (90%)  36 5.2 11

SRS12-34 (96%)  40 5.6 12

SRS12-69 (85%)  58 5.3 13

SRS12-43 (86%)  69 5.3

Synthesis of ethyl 3-(benzylamino)-4-(cyclohexylamino)benzoate(SRS11-92. SI, Table 6)

¹H NMR (CDCl₃, 400 MHz) δ 7.69-7.61 (m, 1H), 7.53-7.30 m, 6H), 6.71-6.62(m, 1H), 4.40-4.28 (m, 4H), 3.97 (b, NH), 3.36 (b, 1H), 3.23 (m, NH),2.17-2.03 (m, 2H), 1.87-1.76 (m, 2H), 1.75-1.66 (m, 1H), 1.47-1.34 (m,5H), 1.32-1.19 (m, 3H); ¹³C NMR (100 MHz, CDCl3) δ 167.3, 142.0, 139.2,134.8, 128.2, 127.44, 123.7, 118.8, 114.7, 109.3, 60.2, 51.4, 49.5,33.3, 25.9, 25.0 14.5; LC/MS (APCI+, M+1) 353.06

Synthesis of ethyl 3-(3-chlorobenzylamino)-4-(cyclohexylamino)benzoate(SRS12-58. SI, Table 6)

¹H NMR (CDCl₃, 400 MHz) δ 7.63 (d, J=4.2 Hz, 1H), 7.43 (s, 2H),7.30-7.28 (m, 3H), 6.65 (d, J=8.4 Hz, 1H), 4.35-4.30 (m, 4H), 3.37 (b,1H), 2.14-2.05 (m, 2H), 1.87-1.76 (m, 2H), 1.75-1.67 (m, 1H), 1.47-1.36(m, 5H), 1.29-1.24 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 167.2, 142.1,141.3, 134.5, 134.4, 129.9, 128.1, 127.6, 126.2, 123.9, 118.9, 115.0,110.0, 60.2, 51.5, 48.9, 33.3, 25.9, 24.9, 14.5; HRMS (FAB) calculatedfor C₂₂H₂₇CIN₂O₂: 386.91; found: 386.18.

Synthesis of ethyl 3-(4-chlorobenzylamino)-4-(cyclohexylamino)benzoate(SRS12-49. SI, Table 6)

¹H NMR (CDCl₃, 400 MHz) δ 7.60 (d, J=8.0 Hz, 1H), 7.41 (s, 1H), 7.34 (s,4H), 6.64 (d, J=8.3 Hz, 1H), 4.36-4.25 (m, 4H), 3.32 (b, 1H), 2.07-2.04(m, 2H), 1.79-1.76 (m, 2H), 1.69-1.33 (5H), 1.26-1.20 (m, 3H); ¹³C NMR(100 MHz, CDCl₃) δ 167.2, 142.0, 137.7, 134.4, 133.2, 129.4, 128.8,123.9, 118.9, 114.9, 109.5, 60.2, 51.4, 48.7, 33.3, 25.9, 24.9 14.5;HRMS (FAB) calculated for C₂₂H2₇CIN₂O₂: 386.91; found: 386.17.

Synthesis of ethyl 3-((4-bromobenzyl)amino)-4-(cyclohexylamino)benzoate(SRS12-35. SI, Table 6)

¹H NMR (CDCl₃, 400 MHz,) δ 7.62 (d, J=8.0 Hz, 1H), 7.50 (d, J=8.4 Hz,2H), 7.42 (s, 1H), 7.31-7.28 (m, 2H), 6.64 (d, J=8.4 Hz, 1H), 4.35-4.28(m, 4H), 3.35 (b, 1H), 2.10-2.07 (m, 2H), 1.81-1.59 (m, 4H), 1.44-1.35(m, 4H), 1.28-1.22 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 167.2, 142.0,138.2, 134.4, 131.7, 129.74, 123.9, 121.2, 118.9, 114.9, 109.5, 60.2,51.4, 48.8, 33.3, 25.9, 24.9, 14.5; HRMS (FAB) calculated forC₂₂H₂₇BrN₂O₂: 431.37; found: 430.13.

Synthesis of ethyl 4-(cyclohexylamino)-3-(3-fluorobenzylamino)benzoate(SRS12-57. SI, Table 6)

¹H NMR (CDCl₃, 400 MHz) δ 7.62 (d, J=8.4 Hz, 1H), 7.43 (s, 1H),7.34-7.31 (m, 1H), 7.21-7.20 (m, 1H), 7.16-7.13 (m, 1H), 7.03-6.99 (m,1H), 6.65 (d, J=8.4 Hz, 1H), 4.35-4.30 (m, 4H), 3.94 (b, NH), 3.56 (b,1H), 3.24 (b, NH), 2.15-2.03 (m, 2H). 1.88-1.75 (m, 2H), 1.76-1.66 (m,1H), 1.49-1.33 (m, 5H). 1.32-1.19 (3H); ¹³C NMR (100 MHz, CDCl₃) δ167.2, 164.3, 161.9, 142.1, 134.4, 130.1, 123.9, 123.5, 123.49, 118.9,115.0, 114.9, 114.7, 114.4, 114.2, 109.5, 60.2, 51.4, 48.9, 33.3, 25.86,24.9, 14.5; ¹⁹F (CDCl₃) δ −112.0; HRMS (FAB) calculated for C₂₂H₂₇FN₂O₂:370.48; found: 370.20.

Synthesis of ethyl 4-(cyclohexylamino)-3-((4-fluorobenzyl)amino)benzoate(SRS12-33. SI, Table 6)

¹H NMR (CDCl₃, 400 MHz) δ 7.62 (d, J=8.4 Hz, 1H), 7.45 (s, 1H),7.41-7.38 (m, 2H), 7.07 (d, J=8.8 Hz, 2H), 6.65 (d, J=8.0 Hz, 1H),4.36-4.288 (m, J=4H), 3.35 (b, 1H), 2.10-2.07 (m, 2H), 1.81-1.61 (m,4H), 1.46-1.44 (m, 4H), 1.39 (t, J=7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl3)δ 167.3, 163.4, 161.0, 142.0, 134.5, 129.7, 123.8, 118.8, 115.4, 114.8,109.4, 60.2, 51.4, 48.7, 33.3, 25.9, 25.0, 14.5; ¹⁹F (CDCl₃) δ −114.3;HRMS (FAB) calculated for C₂₂H₂₇FN₂O₂: 370.46; found: 371.13.

Synthesis of ethyl4-(cyclohexylamino)-3-(4-(trifluoromethyl)benzylamino)-benzoate(SRS12-48. SI, Table 6)

¹H NMR (CDCl₃, 400 MHz) δ 7.61 (td, J=6.2, 2.9 Hz, 3H), 7.52 (d, J=8.0Hz, 2H), 7.40 (d, J=1.9 Hz, 1H), 6.64 (d, J=8.4 Hz, 1H), 4.38 (s, 2H),4.30 (q, J=7.1 Hz, 2H), 3.34 (b, 1H2.11-2.03 (m, 2H), 1.84-1.74 (m, 2H).1.70-1.66 (m, 1H), 1.43-1.32 (m, 5H). 1.2-1.17 (m, 3H); ¹³C NMR (100MHz, CDCl₃) δ 167.2, 143.3, 142.1, 134.2, 128.2, 125.6, 124.0, 118.9,115.0, 109.6, 60.2, 51.4, 50.8, 48.9, 33.3, 25.8, 24.9, 14.4; ¹⁹F(CDCl₃) δ −61.3; HRMS (FAB) calculated for C₂₃H₂₇F₃N₂O₂: 420.47; found:420.22.

Synthesis of ethyl 3-(4-cyanobenzylamino)-4-(cyclohexylamino)benzoate(SRS12-50. SI, Table 6)

¹H NMR (CDCl₃, 400 MHz) δ 7.61 (d, J=2.0 Hz, 2H), 7.59 (d, J=1.6 Hz,1H), 7.50 (d, J=8.4 Hz, 2H), 7.33 (d, J=1.9 Hz, 1H), 6.64 (d, J=8.4 Hz,1H), 4.40 (s, 2H), 4.29 (q, J=7.1 Hz, 2H), 3.34 (b, 1H), 2.09-2.04 (m,2H), 1.77-1.32 (m, 8H), 1.27-1.21 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ167.1, 144.8, 142.1, 133.9, 132.4, 128.4, 124.1, 119.0, 118.8, 115.0,111.2, 110.0, 60.2, 51.4, 48.8, 33.3, 25.9, 24.9, 14.4: HRMS (FAB)calculated for C23H27N3O2: 377.48; found: 377.39.

Synthesis of ethyl 4-(cyclohexylamino)-3-(2-nitrobenzylamino)benzoate(SRS12-71. SI, Table 6)

¹H NMR (CDCl₃, 400 MHz) δ 7.99 (dd, J=8.1, 1.3 Hz, 1H), 7.62-7.58 (m,1H), 7.57-7.54 (m, 1H), 7.52 (d, J=1.5 Hz, 1H), 7.44 (ddd, J=8.1, 7.1,1.7 Hz, 1H), 7.32 (d, J=1.9 Hz, 1H), 6.65-6.58 (m, 1H), 4.58 (s, 2H),4.28 (q, J=7.1 Hz, 2H), 4.08 (s, NH), 3.53 (s, NH), 3.39-3.27 (m, 1H),2.06 (dd, J=12.5, 4.0 Hz, 2H), 2.11-2.02 (m, 2H), 1.82-1.73 (m, 2H),1.71-1.62 (m, 2H), 1.39-1.31 (m, 4H), 1.28-1.22 (m, 3H), 1.27-1.16 (m,3H); ¹³C NMR (100 MHz, CDCl₃) δ 167.1, 149.1, 142.9, 134.5, 133.6,133.3, 131.1, 128.4, 124.9, 124.6, 118.6, 116.6, 109.7, 60.17, 51.42,47.0, 33.2, 25.9, 24.9, 14.4; HRMS (FAB) calculated for C₂₂H₂₇N₃O₄:397.47; found: 397.20.

Synthesis of ethyl 4-(cyclohexylamino)-3-((4-nitrobenzyl)amino)benzoate(SRS12-36. SI, Table 6)

¹H NMR (CDCl₃, 400 MHz) δ 8.24 (d, J=8.4 Hz, 2H), 7.62-7.7.55 (m, 3H),7.34-7.2 (m, 1H), 6.66 (d, 8.4 Hz, 1H), 4.45 (s, 2H), 4.32-4.27 (m, 2H),3.37 (b, 1H), 2.10-2.03 (m, 2H), 1.82-1.67 (m, 4H), 1.44-1.33 (m, 4H),1.29-1.24 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 167.1, 146.8, 142.0,133.8, 129.5, 128.4, 124.1, 123.9, 118.9, 114.9, 109.7, 60.3, 51.5,48.5, 33.3, 25.9, 24.9, 14.5; LC/MS (APCI+, M+1) 397.96

Synthesis of ethyl 4-(cyclohexylamino)-3-((4-methylbenzyl)amino)benzoate(SRS12-34. SI, Table 6)

¹H NMR (CDCl₃, 400 MHz) δ 7.62 (d, J=8.0 Hz, 1H), 7.49 (s, 1H), 7.33 (d,J=8.0 Hz, 2H), 7.20 (d, J=7.6 Hz, 2H), 6.66 (d, J=8.4 Hz, 1H), 4.33 (q,J=6.8, 2H), 4.27 (s, 2H), 3.35 (b, 1H), 2.39 (s, 3H), 2.13-2.0 (m, 2H),1.84-1.74 (m, 2H), 1.73-1.64 (m, 1H), 1.41-1.36 (m, 4H), 1.28-1.21 (m,3H); ¹³C NMR (100 MHz, CDCl₃) δ 167.3, 141.8, 137.1, 136.1, 134.8,129.3, 128.2, 123.6, 118.9, 114.7, 109.3, 60.2, 51.5, 49.25, 33.3, 25.9,25.0, 21.1, 14.5; HRMS (FAB) calculated for C₂₃H₃₀N₂O₂: 366.50; found:366.23.

Synthesis of ethyl4-(cyclohexylamino)-3-(4-(methoxycarbonyl)-benzylamino)benzoate(SRS12-69. SI, Table 6)

¹H NMR (CDCl₃, 400 MHz) δ 7.60 (ddd, J=8.4, 1.9, 1.0 Hz, 1H), 7.45 (d,J=1.8 Hz, 1H), 7.32-7.27 (m, 1H), 7.04-6.95 (m, 2H), 6.89-6.81 (m, 1H),6.62 (dd, J=8.7, 1.3 Hz, 1H), 4.31 (tdd, J=7.2, 6.7, 1.2 Hz, 2H), 4.27(s, 2H), 3.90 (s, NH), 3.82 (d, J=1.1 Hz, 3H), 3.33 (s, 1H), 2.09-2.02(m, 2H), 1.82-1.73 (m, 2H), 1.71-1.63 (m, 1H), 1.45-1.32 (m, 5H),1.26-1.17 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 167.3, 159.9, 141.9,140.8, 134.7, 129.7, 123.7, 120.5, 118.9, 114.8, 113.8, 112.8, 109.4,60.2, 55.2, 49.5, 33.3, 25.9, 25.0, 14.5; HRMS (FAB) calculated forC₂₃H₃₀N₂O₃: 382.50; found: 382.37.

Synthesis of ethyl4-(cyclohexylamino)-3-((4-methoxybenzyl)amino)-benzoate (SRS12-43. SI,Table 6)

¹H NMR (CDCl₃, 400 MHz) δ 7.63 (d, J=8.4 Hz, 1H), 7.49 (s, 1H), 7.37 (d,J=8.4 Hz, 2H), 6.94 (d, J=8.8 Hz, 2H), 6.65 (d, J=8.4 Hz, 1H), 4.5 (q,J=7.2, 2H), 4.26 (s, 2H), 3.85 (s, 3H), 3.35 (b, 1H), 2.10-2.07 (m, 2H),1.82-1.60 (m, 4H), 1.47-1.33 (m, 4H), 1.30-1.27 (m, 3H); LC/MS (APCI+,M+1) 282.19.

TABLE 7 SI, EC50 of Ferrostatin-1 analogs.

Name EC50 Log Entry R₁ R₂ (Yield) (nM) P  1

H SRS11-92 (98%)   6 5.3  2 R_(1a) tert- butyl SRS13-29 (95%)  83 6.8  3

H SRS12-51 (91%)  58 6.5  4

H SRS12-46 (85%)  33 4.0  5

H SRS13-12 (87%)  48 4.0  6

H SRS12-45 (90%)  25 4.0  7 R_(1b) tert- butyl SRS13-30 (89%) 114 5.6  8

H SRS13-35 (92%)  27 2.9  9 R_(1c) tert- butyl SRS13-37 (88%)  15 4.4 10

H SRS12-54 (85%) 159 5.1 11

H SRS12-59 (86%)  96 5.7 12

H SRS12-52 (89%) 105 6.3 13

H SRS12-53 (85%)  41 5.3 14

H 4MO43 (85%)  47 5.2 15

H SRS12-80; R₂ = CH₃ (86%)  33 5.2 16 R_(1d) H SRS12-84; R₂ = CH₂CH₃(88%)  53 5.6

Synthesis of single isomer of ethyl3-(benzylamino)-4-(4-tert-butylcyclohexylamino)benzoate (SRS13-29. SI,Table 7)

¹H NMR (CDCl₃, 400 MHz) δ 7.62 (d, J=8.3, 1H), 7.48-7.32 (m, 6H), 6.65(d, J=8.3 Hz, 1H), 4.35-4.29 (m, 4H), 3.36 (b, 1H), 2.19 (s, 2H), 1.86(s, 2H), 1.38 (t, J=7.1, 0.8 Hz, 3H), 1.22-1.15 (m, 4H), 1.09-1.03 (m,1H), 0.90 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 167.3, 141.9, 139.2,134.8, 128.7, 128.3, 127.5, 123.6, 118.8, 114.5, 109.3, 60.2, 52.1,49.5, 47.6, 33.8, 32.4, 27.6, 26.2, 14.5; HRMS (FAB) calculated forC₂₆H₃₆N₂O₂: 408.58; found: 408.28.

Synthesis of ethyl4-(cyclohexylamino)-3-(naphthalen-2-ylmethylamino)-benzoate (SRS12-51.SI, Table 7)

¹H NMR (CDCl₃, 400 MHz) δ 7.87-7.82 (m, 4H), 7.63-7.60 (m, 1H), 7.55 (d,J=5.0 Hz, 1H), 7.51 (s, 1H), 7.50-7.48 (m, 2H), 6.64 (d, J=8.4 Hz, 1H),4.46 (s, 2H), 4.31 (q, J=7.1 Hz, 2H), 3.93 (s, NH,) 3.34 (b, 1H), 3.24(b, NH), 2.09-2.03 (m, 2H), 1.79-1.63 (m, 3H), 1.45-1.33 (m, 5H),1.27-1.19 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 167.3, 142.0, 136.7,134.8, 133.5, 132.86, 128.4, 127.8, 127.7, 126.7, 126.4, 126.2, 125.9,123.8, 118.9, 114.8, 109.4, 60.2, 51.4, 49.7, 33.3, 25.9, 25.0, 14.5;HRMS (FAB) calculated for C₂₆H₃₀N₂O₂ 402.53; found: 402.23.

Synthesis of ethyl4-(cyclohexylamino)-3-(pyridin-2-ylmethylamino)-benzoate (SRS12-46. SI,Table 7)

¹H NMR (CDCl₃, 400 MHz) δ 8.62 (d, J=4.4 Hz, 1H), 7.71-7.67 (m, 1H),7.61-7.59 (m, 1H), 7.42 (s, 1H), 7.34 (d, J=8.0 Hz, 1H), 7.28 (s, 1H),7.24-7.21 (m, 1H), 6.64 (d, J=8.4 Hz, 1H), 4.45 (s, 2H), 4.32 (q, J=7.2,2H), 3.36 (b, 1H), 2.12-2.09 (m, 2H), 1.85-1.66 (m, 3H), 1.46-1.34 (m,5H), 1.32-1.24 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 167.3, 158.1, 149.2,142.1, 136.7, 134.6, 123.6, 122.3, 122.2, 118.6, 114.7, 109.2, 51.4,50.1, 33.3, 26.1, 25.9, 25.0, 14.5; HRMS (FAB) calculated forO₂₁H₂₇N₃O₂: 353.46; found: 353.21.

Synthesis of ethyl4-(cyclohexylamino)-3-(pyridin-3-ylmethylamino)-benzoate (SRS13-12. SI,Table 7)

¹H NMR (CDCl₃, 400 MHz) δ 8.70 (d, J=2.3 Hz, 1H), 8.59-8.56 (m, 1H),7.75 (d, J=7.6 Hz, 1H), 7.64-7.53 (m, 1H), 7.44 (s, 1H), 7.37-7.26 (m,1H), 6.66 (d, J=8.8 Hz, 1H), 4.36-4.31 (m, 4H) 3.39-3.33 (b, 1H),2.13-2.01 (m, 2H), 1.87-1.75 (m, 2H), 1.71-1.66 (m, 1H), 1.43-1.33 (m,5H), 1.30-1.20 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 167.2, 149.3, 148.6,136.1, 134.0, 124.18, 123.7, 115.1, 112.3, 109.6, 60.3, 51.5, 46.9,33.29, 31.3, 25.83, 24.9, 14.5; HRMS (FAB) calculated for C₂₁H₂₇N₃O₂:353.46; found: 354.12.

Synthesis of ethyl4-(cyclohexylamino)-3-((pyridin-4-ylmethyl)amino)-benzoate (SRS12-45.SI, Table 7)

¹H NMR (CDCl₃, 400 MHz) δ 8.50 (d, J=1.2 Hz, 1H), 7.62 (d, J=7.6 Hz,1H), 7.35 (s, 1H), 6.66 (d, J=8.4 Hz, 1H), 4.37-4.28 (m, 4H), 3.36 (b,1H), 2.11-2.08 (m, 2H), 1.84-1.76 (m, 2H), 1.76-1.70 (m, 1H), 1.44-1.33(m, 4H), 1.29-1.22 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 167.08, 150.04,148.31, 142.06, 133.98, 124.05, 122.63, 118.98, 115.01, 109.73, 60.22,51.44, 48.06, 33.34, 25.85, 24.91, 14.44: HRMS (FAB) calculated forO₂₁H₂₇N₃O₂: 353.46; found: 354.21.

Synthesis of ethyl4-(4-tert-butylcyclohexylamino)-3-(pyridin-4-ylmethylamino)benzoate(SRS13-30. SI, Table 7)

¹H NMR (CDCl₃, 400 MHz) δ 8.63-8.55 (m, 2H), 7.62 (dd, J=8.4, 1.9 Hz,1H). 7.38-7.30 (m, 3H), 6.67 (d, J=8.4 Hz, 1H), 4.37 (s, 2H), 4.31 (q,J=7.1 Hz, 2H), 3.30-3.24 (m, 1H), 2.28-2.16 (m, 2H), 1.93-1.82 (m, 2H),1.36 (t, J=7.1 Hz, 3H), 1.24-1.16 (m, 4H,), 1.11-1.05 (m, 1H), 0.91 (s,9H); ¹³C NMR (100 MHz, CDCl₃) δ 167.1, 150.0, 148.3, 142.2, 134.0,124.0, 122.6, 119.0, 114.9, 109.8, 60.2, 52.1, 48.1, 47.7, 33.9, 32.4,27.6, 26.2, 14.4; HRMS (FAB) calculated for C₂₅H₃₅N₃O₂: 409.56; found:409.27.

Synthesis of ethyl4-(cyclohexylamino)-3-(pyrimidin-5-ylmethylamino)-benzoate (SRS13-35.SI, Table 7)

¹H NMR (CDCl₃, 400 MHz) δ 9.17 (s, 1H), 8.63 (d, J=5.1 Hz, 1H), 7.57 (d,J=8.3 Hz, 1H), 7.34-7.27 (m, 2H), 6.62 (d, J=8.4 Hz, 1H), 4.41 (s, 2H),4.26 (q, J=7.1 Hz, 2H), 4.15 (s, 2H), 3.34 (b, 1H), 2.07 (d, J=11.8 Hz,2H), 1.77 (d, J=13.1 Hz, 2H), 1.66 (d, J=12.1 Hz, 1H), 1.39-1.21 (m,8H); ¹³C NMR (100 MHz, CDCl₃) δ 167.1, 158.6, 157.0, 141.9, 133.8,123.8, 119.3, 118.7, 114.4, 109.5, 60.2, 51.4, 49.3, 33.2, 25.8, 24.9,14.5; LC/MS (APCI+, M+1) 354.69

Synthesis of ethyl4-(4-tert-butylcyclohexylamino)-3-(pyrimidin-5-ylmethylamino)benzoate(SRS13-37. SI, Table 7)

¹H NMR (CDCl₃, 400 MHz) δ 9.23 (s, 1H), 8.74-8.64 (m, 1H), 7.62 (s, 1H),7.41-7.31 (m, 2H), 6.66 (d, J=8.4 Hz, 1H), 4.47 (s, 2H), 4.31 (d, J=10.0Hz, 2H), 4.04 (d, J=16.2 Hz, 2H), 2.23 (s, 2H), 1.89 (s, 2H), 1.35 (t,J=7.1 Hz, 3H), 1.21 (s, 4H), 1.09 (d, J=9.0 Hz, 1H), 0.92-0.088 (s, 9H);¹³C NMR (100 MHz, CDCl₃) δ 166.9, 158.7, 157.1, 142.1, 133.8, 123.9,119.3, 118.8, 114.6, 109.6, 60.2, 52.1, 49.3, 47.7, 33.8, 32.4, 27.6,26.2, 14.5; LC/MS (APCI+, M+1) 410.19

Synthesis of ethyl3-(3-cyano-4-fluorobenzylamino)-4-(cyclohexylamino)-benzoate (SRS12-54.SI, Table 7)

¹H NMR (CDCl₃, 400 MHz) δ 7.70-7.65 (m, 1H), 7.62-7.60 (m, 2H), 7.31 (s,1H), 7.20 (t, J=8.8 Hz, 1H), 6.65 (d, J=8.4 Hz, 1H), 4.34-4.27 (m, 4H),3.35 (b, 1H), 2.10-2.04 (m, 2H), 1.83-1.75 (m, 2H), 1.72-1.65 (m, 1H),1.45-1.31 (m, 5H), 1.29-1.19 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 167.1,163.7, 161.1, 142.1, 136.4, 134.4, 133.6, 132.5, 124.2, 118.9, 116.5,115.0, 113.9, 109.8, 60.3, 51.5, 47.8, 33.4, 25.84, 24.9, 14.4; ¹⁹F(CDCl₃) δ −107.7; HRMS (FAB) calculated for C₂₃H₂₆FN₃O₂: 395.47; found:395.08.

Synthesis of ethyl4-(cyclohexylamino)-3-(3,5-difluorobenzylamino)-benzoate (SRS12-59. SI,Table 7)

¹H NMR (CDCl₃, 400 MHz) δ 7.62 (d, J=8.0 Hz, 1H), 7.37 (s, 1H),7.30-6.95 (d, J=6.8 Hz, 2H), 6.76-6.72 (m, 1H), 6.65 (d, J=8.4 Hz, 1H),4.32-4.30 (m, 4H), 3.93 (b, NH), 3.36 (b, 1H), 3.28 (b, NH), 2.11-2.08(m, 2H), 1.82-1.79 (m, 2H), 1.71-1.69 (m, 1H), 1.47-1.33 (m, 5H),1.31-1.20 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 167.1, 164.5, 162.0,143.4, 142.1, 134.0, 124.1, 118.9, 115.0, 110.5, 109.7, 102.7, 60.2,51.4, 48.6, 33.3, 25.86, 24.9, 14.4; ¹⁹F (CDCl₃) δ −108.7; HRMS (FAB)calculated for C₂₂H₂₆F₂N₂O₂: 388.45; found: 388.00.

Synthesis of ethyl3-(3-bromo-5-fluorobenzylamino)-4-(cyclohexylamino)-benzoate (SRS12-52.SI, Table 7)

¹H NMR (CDCl₃, 400 MHz) δ 7.60 7.61 (dd, J=8.4, 1.9 Hz, 1H), 7.35 (d,J=1.8 Hz, 2H), 7.17 (dt, J=8.1, 2.1 Hz, 1H), 7.06 (d, J=9.2 Hz, 1H),6.64 (d, J=8.4 Hz, 1H), 4.33-4.27 (m, 4H), 3.91 (s, NH), 3.34 (b, 1H),3.24 (s, NH), 2.12-2.03 (m, 2H), 1.84-1.75 (m, 2H), 1.72-1.65 (m, 1H),1.45-1.32 (m, 5H), 1.29-1.21 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 167.1,164.1, 161.6, 143.5, 142.1, 134.0, 126.7, 124.1, 122.7, 118.2, 115.1,113.8, 109.7, 60.2, 51.5, 48.4, 33.3, 25.9, 24.9, 14.4; ¹⁹F (CDCl₃)−109.6; HRMS (FAB) calculated for C22H₂₆BrFN₂O₂: 449.36; found: 450.00.

Synthesis of ethyl4-(cyclohexylamino)-3-(3,5-dimethoxybenzylamino)-benzoate (SRS12-53. SI,Table 7)

¹H NMR (CDCl₃, 400 MHz) δ 7.60 (dd, J=8.3, 1.9 Hz, 1H), 7.44 (d, J=2.0Hz, 1H), 6.62 (d, J=8.4 Hz, 1H), 6.58 (d, J=2.3 Hz, 2H), 6.42-6.4 (m,1H), 4.31 (q, J=7.1 Hz, 2H), 4.23 (s, 2H), 3.80 (s, 6H), 3.33 (b, 1H),2.07-2.04 (m, 2H), 1.79-1.34 (m, 8H), 1.26-1.20 (m, 3H); LC/MS (APCI+,M+1) 412.67

Synthesis of ethyl4-(cyclohexylamino)-3-(4-(ethanoyloxy)benzylamino)-benzoate (4M043. SI,Table 7)

¹H NMR (400 MHz, CDCl₃) δ 7.61 (dd, J=8.3, 1.9 Hz, 1H), 7.46-7.40 (m,3H), 7.09 (d, J=8.5 Hz, 2H), 6.63 (d, J=8.5 Hz, 1H), 4.37-4.29 (m, 2H),4.28 (s, 2H), 3.33 (s, 1H), 2.31 (s, 3H), 2.10-2.03 (m, 2H), 1.78 (d,J=13.4 Hz, 2H), 1.67 (d, J=12.8 Hz, 1H), 1.41 (s, 1H), 1.36 (t, J=7.1Hz, 4H), 1.27-1.19 (m, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 169.6, 167.3,150.0, 142.0, 136.8, 134.61, 129.4, 123.8, 121.8, 118.8, 114.6, 109.3,60.2, 51.5, 48.9, 33.4, 25.9, 25.0, 21.1, 14.5; LC/MS (APCI+, M+1)411.01

Synthesis of ethyl4-(cyclohexylamino)-3-(4-(methoxycarbonyl)-benzylamino)benzoate(SRS12-80. SI, Table 7)

¹H NMR (CDCl₃, 400 MHz) δ 8.07-7.99 (m, 2H), 7.60 (dd, J=8.4, 1.9 Hz,1H), 7.51-7.44 (m, 2H), 7.40 (d, J=1.9 Hz, 1H), 6.64 (d, J=8.4 Hz, 1H),4.37 (s, 2H), 4.30 (q, J=7.1 Hz, 2H), 3.92 (d, J=0.6 Hz, 3H), 3.89 (d,J=0.7 Hz, 1H), 3.39-3.29 (m, 1H), 2.11-2.02 (m, 2H), 1.83-1.73 (m, 2H),1.72-1.62 (m, 1H), 1.48-1.30 (m, 5H), 1.30-1.16 (m, 3H); ¹³C NMR (100MHz, CDCl₃) δ 167.2, 166.9, 144.5, 142.0, 134.3, 130.0, 128.7, 127.8,123.9, 118.9, 114.9, 109.5, 60.2, 52.1, 51.4, 49.0, 33.3, 25.9, 24.9,14.5; HRMS (FAB) calculated for C₂₄H₃₀N₂O₄: 410.51; found: 410.10.

Synthesis of ethyl4-(cyclohexylamino)-3-(4-(ethoxycarbonyl)benzylamino)-benzoate(SRS12-84. SI, Table 7)

¹H NMR (CDCl₃, 400 MHz) δ 8.05 (d, J=8.3 Hz, 2H), 7.61 (dd, J=8.3, 1.9Hz, 1H), 7.48 (d, J=8.3 Hz, 2H), 7.41 (d, J=1.8 Hz, 1H), 6.66-6.62 (m,1H), 4.41-4.36 (m, 4H), 4.30 (t, J=7.1 Hz, 2H), 3.35 (s, 1H), 3.11 (d,J=7.5 Hz, 1H), 2.07 (d, J=12.8 Hz, 2H), 1.79 (d, J=13.4 Hz, 2H), 1.68(d, J=13.0 Hz, 1H), 1.44-1.36 (m, 8H), 1.26 (d, J=7.9 Hz, 3H); ¹³C NMR(100 MHz, CDCl₃) δ 167.2, 166.4, 144.3, 142.0, 134.3, 129.9, 129.6,128.7, 127.8, 123.9, 118.9, 114.9, 109.5, 60.9, 60.2, 57.8, 51.4, 49.0,33.3, 25.9, 24.9, 14.5; HRMS (FAB) calculated for C₂₅H₃₂N₂O₄: 424.53;found: 424.12.

TABLE 8 SI, EC50 of Ferrostatin-1 analogs.

Entry R1 R₂ R₃ Name (Yield) EC50 (nM) Log P 1 Ferrostatin-1

NH₂

SRS8-28 (85%) 88 3.2 2

NO₂

SRS8-24 (77%) >10,000 3.8 3 Cl NH₂

SRS8-62 (89%) >10,000 2.1 4 H NH₂

CA₁ >10,000 1.6 5 NH₂ NH₂

CA₂ 6900 0.5

Synthesis of ethyl 5-amino-6-(cyclohexylamino)pyridine-3-carboxylate(SRS14-86. SI, Scheme 2)

Following the above general procedure A and B and starting from theethyl ester (SRS14-84, SI, Scheme 2), which was prepared from thecorresponding acid, the crude reaction mixture was purified by columnchromatography (dichloromethane: methanol=20:1) to provide the desiredethyl 5-amino-6-(cyclohexylamino)pyridine-3-carboxylate (SRS14-86, SI,Scheme 2) (195 mg, 0.739 mmol, 85% (2 steps)). ¹H NMR (400 MHz, CDCl₃) δ8.42 (d, J=2.0 Hz, 1H), 7.36 (d, J=2.0 Hz, 1H), 4.78 (d, J=7.6 Hz, 1H),4.30-4.21 (m, 2H), 4.08-3.92 (m, 1H), 3.36 (s, 1H), 2.07-1.56 (m, 10H),1.42-1.24 (m, 3H); LC/MS (APCI+, M+1) 264.26.

Synthesis of ethyl5-(benzylamino)-6-(cyclohexylamino)pyridine-3-carboxylate (SRS14-91. SI,Scheme 2)

Following the above general procedure C or D, the crude reaction mixturewas purified by column chromatography (dichloromethane: methanol=40:1)to provide the desired ethyl 5-(benzylamino)-6(cyclohexylamino)-pyridine-3-carboxylate (SRS14-91, SI, Scheme 2) (14.8mg, 0.04 mmol, 53%). ¹H NMR (500 MHz, CDCl₃) δ 8.49 (d, J=1.9 Hz, 1H),7.40 (dd, J=34.8, 27.7 Hz, 5H), 4.59 (s, 1H), 4.47-4.25 (m, 4H), 4.08(s, 1H), 2.11-1.51 (m, 10H), 1.38 (t, J=11.5, 4.4 Hz, 3H); LC/MS (APCI+,M+1) 354.66.

Synthesis of ethyl6-(cyclohexylamino)-5-(pyridin-4-ylmethylamino)pyridine-3-carboxylate(SRS14-92. SI, Scheme 2)

Following the above general procedure C or D, the crude reaction mixturewas purified by column chromatography (dichloromethane: methanol=20:1)to provide the desired ethyl6-(cyclohexylamino)-5-(pyridin-4-ylmethylamino)-pyridine-3-carboxylate(SRS14-92, SI, Scheme 2) (16 mg, 0.045 mmol, 54%), ¹H NMR (400 MHz,CDCl₃) δ 8.55 (d, J=50.9 Hz, 3H), 7.46-7.15 (m, 3H), 4.67 (s, 1H),4.40-4.25 (m, 2H), 4.07 (s, 2H), 2.10-1.62 (m, 10H), 1.40-1.19 (m, 3H);LC/MS (APCI+, M+1) 355.36.

Synthesis of ethyl 6-(cyclohexylamino)-5-(pyrimidin-4ylmethylamino)-pyridine-3-carboxylate (SRS14-93. SI, Scheme 2)

Following the above general procedure C or D, the crude reaction mixturewas purified by column chromatography (dichloromethane: methanol=15:1)to provide the desired ethyl6-(cyclohexylamino)-5-(pyrimidin-4-yl-methylamino)-pyridine-3-carboxylate(SRS14-93, SI, Scheme 2), (18 mg, 0.051 mmol, 58%).

¹H NMR (500 MHz, CDCl₃) δ 9.26 (s, 1H), 8.74 (d, J=5.1 Hz, 1H), 8.49 (d,J=1.8 Hz, 1H), 7.37 (d, J=5.1 Hz, 1H), 7.32 (d, J=1.8 Hz, 1H), 4.40 (d,J=40.5 Hz, 2H), 4.34 (dd, J=14.2, 7.1 Hz, 2H), 4.11 (s, 1H), 2.11-1.48(m, 10H), 1.42-1.31 (m, 3H); LC/MS (APCI+, M+1) 356.26.

Synthesis of ethyl 5-amino-4-(cyclohexylamino)-2-fluorobenzoate(SRS14-55. SI, Scheme 3)

Following the above general procedure A and B and starting from theethyl ester (SRS14-41, SI, Scheme 3), which was prepared from thecorresponding acid, the crude reaction mixture was purified by columnchromatography (dichloromethane: methanol=20:1) to provide the desiredethyl 5-amino-4-(cyclohexylamino)-2-fluorobenzoate (SRS14-55, SI, Scheme3) (109 mg, 0.389 mmol, 90% (2 steps)). ¹H NMR (400 MHz, CDCl₃) δ7.37-7.26 (m, 1H), 6.36-6.23 (m, 1H), 4.34 (dd, J=8.9, 6.2, 1.8 Hz, 2H),3.27 (s, 1H), 3.01 (s, 1H), 2.07 (d, J=8.6 Hz, 2H), 1.81 (d, J=8.5 Hz,2H), 1.69 (s, 1H), 1.38 (ddd, J=8.9, 6.3, 3.1 Hz, 5H), 1.27 (s, 3H);LC/MS (APCI+, M+1) 281.36.

Synthesis of ethyl 5-amino-2-chloro-4-(cyclohexylamino)benzoate(SRS14-57. SI, Scheme 4)

Following the above general procedure A and B and starting from theethyl ester (SRS14-40, SI, Scheme 4), which was prepared from thecorresponding acid, the crude reaction mixture was purified by columnchromatography (dichloromethane: methanol=20:1) to provide the desiredethyl 5-amino-2-chloro-4-(cyclohexylamino)benzoate (SRS14-57, SI, Scheme4) (136 mg, 0.459 mmol, 75% (2 steps)). ¹H NMR (400 MHz, CDCl₃) δ 7.57(d, J=8.4 Hz, 1H), 7.42 (s, 1H), 6.59 (d, J=8.4 Hz, 1H), 4.17-4.06 (m,2H), 3.47 (s, 1H), 3.35-3.27 (m, 1H), 1.78 (d, J=13.0 Hz, 2H), 1.67 (d,J=12.0 Hz, 1H), 1.42-1.31 (m, 5H), 1.26 (td, J=7.1, 1.6 Hz, 5H); LC/MS(APCI+, M+1) 297.47.

Synthesis of ethyl 5-amino-4-(cyclohexylamino)-2-methylbenzoate(SRS14-58. SI, Scheme 5)

Following the above general procedure A and B and starting from theethyl ester (SRS14-43, SI, Scheme 5), which was prepared from thecorresponding acid, the crude reaction mixture was purified by columnchromatography (dichloromethane: methanol=40:1) to provide the desiredethyl 5-amino-4-(cyclohexylamino)-2-methylbenzoate (SRS14-58, SI, Scheme5) (18 mg, 0.064 mmol, 83% (2 steps)). ¹H NMR (400 MHz, CDCl₃) δ 7.43(d, J=2.2 Hz, 1H), 6.41 (s, 1H), 4.30 (dd, J=9.5, 4.6 Hz, 2H), 3.40-3.28(m, 2H), 2.56 (d, J=2.6 Hz, 3H), 2.09 (d, J=9.3 Hz, 2H), 1.81 (d, J=10.0Hz, 2H), 1.70 (d, J=8.6 Hz, 1H), 1.39 (ddd, J=14.9, 9.3, 8.1 Hz, 5H),1.29-1.16 (m, 3H); LC/MS (APCI+, M+1) 277.16.

Results

The 67 analogs were tested for cell death inhibition by treating HT-1080fibrosarcoma cells with a lethal concentration of erastin (10 μM) in thepresence of each ferrostatin analog. Based on the activity of theanalogs generated in this assay, a structure activity relationship (SAR)for the Fer-1 scaffold (FIG. 17) was established. Analogs with a nitrogroup in place of the amine were inactive, confirming previoussuggestions that this amine is essential for ferrostatin activity. Inaddition, analogs lacking cyclohexylamines were not active. Introductionof heteroatoms into the N-cyclohexyl moiety resulted in consistentreductions in potency, consistent with the hypothesis that thehydrophobicity of this portion of the scaffold is crucial for anchoringor concentrating within lipid membranes. Modifications of the ethylester, some involving substantial extensions, were generally welltolerated. Finally, both amines are essential for full activity and theamines must be mono-substituted or unsubstituted; the sole exceptionbeing the imine of SRS16-86, which can perhaps be reduced in cells.Thus, it appears that the ability to oxidize both amines is crucial forthe potency of ferrostatins (Scheme 6). In addition, conversion of theamine group into methyl-, benzyl- or tert-butylcarbamates or amides wasnot tolerated, consistent with the mechanism proposed in Scheme 6, asthese groups will interfere with the tautomerization, which will resultin the inhibition of the release of 2 protons and 2 electrons. Arylamine substituents with electron withdrawing or electron donating groups(—Cl, —Br, —F, —CN, —CF₃, —NO₂, esters or —OCH₃) or nitrogen in thearomatic ring did not affect potency, suggesting that thesemodifications did not inhibit the electronic delocalization and therelease of reducing equivalents into the cell. Overall, this series ofFer-1 analogs provided insight into the Fer-1 mechanism of action andalso allowed the identification of analogs with greater potency andimproved properties. Indeed, a number of compounds were discovered(SRS11-92, SRS12-45, SRS13-35 and SRS13-37) that were more potent thanFer-1 (FIG. 18A; also discussed in International Application Serial No.PCT/US/2013/035021, filed Apr. 2, 2013, which is incorporated byreference in full herein). For example, the SRS11-92 was 15-fold morepotent than the parent Fer-1, with an EC50=6 nM (FIG. 18a , entry 3). AnX-ray structure of SRS11-92 analog was obtained (FIG. 18B) to confirmits purity and the orientation of both secondary amines.

In parallel with the above cell-based assay, a2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was used to examine theability of each analog to scavenge free radicals in a cell-free in vitrosolution—a test of intrinsic antioxidant capacity. The reduction of theDPPH radical by ferrostatins resulted in a decrease in absorbance,indicating the antiradical capacity of the ferrostatins. Redox activeanalogs scavenged the DPPH radical by 60-90% within 30 minutes (FIG. 18a). Consistent with the notion that the lipophilic character of theN-cyclohexyl ring is essential for cell death inhibition in cells, butnot intrinsic anti-oxidant activity, Fer-1 analogs bearing heteroatomsubstitutions such as O- or N-methyl in this moiety retained antioxidantactivity in the DPPH assay (68% and 86% reduction of DPPH respectively,despite resulting in much lower potency in the HT-1080 death-suppressionassay. Conversely, analogs with electron-withdrawing functional groups(i.e. carbamate and amide analogs) on the primary amine did not scavengethe DPPH radical (0-10%) and did not prevent cell death, suggesting thatthese groups act to prevent the delocalization and oxidation necessaryfor radical scavenging, as proposed in Scheme 6.

As a first step in investigating inhibition of ferroptosis in vivo,select ferrostatin analogs were tested for their stability in mousemicrosomes and plasma. These compounds had half-lives (T_(1/2))<12 min,where a T_(1/2)>30 is generally accepted as indicative of minimallyacceptable in vivo PK. Interestingly, an imine analog (SRS16-80) of oneof the most potent analogs, incorporating an adamantyl ring in place ofthe cyclohexyl moiety, showed a T_(1/2) of 154 min. While the imine inSRS16-80 led to about 6-fold lower potency (EC50˜300 nM), itcorroborated the hypothesis that the benzylic position was the source ofcytochrome P450 reactivity. Interestingly, in silico evaluation of theseFer-1 analogs' microsmal stability (Schrodinger Suite P450_SOM) was inagreement with the experimental results.

The imine was then evaluated for mouse plasma stability and found to berapidly hydrolyzed, due to an isopropyl ester present in this compound(T_(1/2)<15 min). This plasma instability was prevented by replacing theisopropyl ester with a t-butyl ester, creating a compound (SRS16-86)with a T_(1/2)>120 min in plasma and in liver microsomes (EC50˜350 nM).

Example 16 Design and Synthesis of Microsome and Plasma StableFerrostatin Analogs

Experimental data pointed to the benzylic position of ferrostatinanalogs as the site of metabolic liability in microsomes, and the estergroup as the target of plasma esterases. Therefore, analog synthesisfocuses on modification of these positions with the goal of improvingmicrosomal and plasma stability in vitro and with the ultimate goal ofproducing analogs with improved in vivo properties for use in animalmodels of disease. Because in silico evaluation of Fer-1 analogs' P450stability using the Schrodinger Suite P450_SOM program showed agreementwith the experimental results with liver microsomes, this computerprogram is used to guide prioritization of compound synthesis andtesting of analogs proposed based on modifications known to inhibitmetabolism.

One of the most useful methods of blocking metabolism at a specific siteis to use a steric shield—a bulky group that hinders oxidation at theposition by cytochrome P450. An efficient synthesis of Fer-1 analogswith bulky, blocking groups incorporated at the benzylic site ofoxidation (FIG. 19) is shown in Scheme 7.

This straightforward synthetic route is a variation of the synthesisoutlined in Scheme 5. Treatment of commercially available3-fluoro-4-nitrobenzoic acid with a benzylamine containing the desiredbulky substituent at the benzylic position would displace fluoride viaan SNAr reaction to give the corresponding aminonitro compound (Saitoh,et al., 2009). A wide range of benzyl amines are commercially available,including the enantiomerically pure ones shown in FIG. 19.Enantiomerically pure amines are important because cytochrome P450s areknown to be enantioselective in their oxidations. Benzylicallydisubstituted amines would increase the amount of steric shielding andhave the advantage of being achiral. The 2,6-dimethylbenzyl amineillustrates another mode of shielding the benzylic position.

The synthetic route shown in Scheme 7 also allows ready access to othersubstituted amine analogs that can be explored, and that may be moreresistant to metabolism, as they do not have a benzylic position toreact with P450s. Thus, aniline, cyclohexylamine, and adamantly aminemay be used as starting materials to give the corresponding analogs(FIG. 19).

The t-butyl ester is resistant to plasma esterases; however, this groupmay be acid labile, and may not be resistant to the acidic conditions inthe stomach upon oral dosing. Bioisosteres, functionalities that arebiologically equivalent to the functional group they are replacing, arecommonly used to produce active analogs with improved properties, suchas resistance to metabolism (Hamada, et al., 2012). A number of esterbioisosteres have been reported in the literature and can beincorporated into analogs of Fer-1. As shown in the synthetic route inScheme 8, the acid or ester group of 3-fluoro-4-nitrobenzoic acid can bereadily converted into ester bioisosteres, such as oxazoles (Wu, et al.,2004), oxadiazoles (Pipik, et al., 2004), triazoles (Passaniti, et al.,2002), or ketones (Genna, et al., 2011). These intermediates can then beused in the synthetic route outlined in Scheme 7 to produce the desiredFer-1 analogs with ester bioisosteres that are resistant to esterases(FIG. 20).

All analogs are tested in vitro for their ability to inhibiterastin-induced ferroptosis in cells. Those with an IC₅₀ of <50 nM aretested for metabolic stability in mouse liver microsomes and plasma.Those analogs with T_(1/2)>30 minutes in those assays undergopharmacokinetic analysis in mice. Those analogs with the best in vivo PKparameters are tested in the HD mouse model (see below).

Rescue Activity of Fer-1 Analogs (Dixon, et al., 2012)

HT-1080 cells are cultured in DMEM containing 10% fetal bovine serum, 1%supplemented non-essential amino acids and 1% pen/strep mixture (Gibco)and maintained in a humidified environment at 37° C. with 5% CO₂ in atissue culture incubator. 1,000 HT-1080 cells are seeded per well induplicate 384-well plates (Corning) using a BioMek FX liquid handlingrobot (Beckman Coulter). The next day, the medium is replaced with 36 μLof medium containing 10 μM erastin with 4 μL of medium containing adilution series (previously prepared) of DMSO, Fer-1 (positive control)or Fer-1 analogs. 24 hours later, 10 μL Alamar Blue (Invitrogen) cellviability solution is added to the growth media to a final concentrationof 10%. Cells are incubated a further 6 hours and then the Alamar Bluefluorescence intensity recorded using a Victor 3 platereader(PerkinElmer)(ex/em 530/590). All experiments are performed at leasttwice and the background (no cells)-subtracted Alamar Blue values foreach combination are averaged between replicates. From these data,sigmoidal dose-response viability curves and EC50 values are computedusing Prism 5.0 (GraphPad).

Plasma and Metabolic Stability

Each compound (1 μM) is incubated with mouse plasma, for 4 hours at 37°C., with shaking at 100 rpm. The concentration of compound in the bufferand plasma chambers is determined using LC-MS/MS. Metabolism of eachcompound is predicted using Sites of Metabolism (Schrodinger Suite),which combines intrinsic reactivity analysis (Hammett-Taft) with inducedfit docking against 2C9, 2D6 and 3A4. This approach identifies 90% ofknown metabolism sites and has a false positive rate of 17%. The invitro metabolic stability of each compound in mouse liver microsomes isdetermined. Pooled mouse liver microsomes are prepared and stored at−80° C. until needed. Compound stability in liver microsomes is measuredat 0, 15, 30, 45 and 60 minutes in duplicate, using LC-MS/MS analysis.

Pharmacokinetic Evaluation of Compounds in Mice

To evaluate the PK profile of compounds, IV, IP, and PO administrationof each compound is used in C57BL/6J wt mice. Mice are dosed IV at 10mg/kg and sacrificed using Nembutal and CO₂ euthanasia. Six week oldmice (Charles River) that have been acclimated to their environment for2 weeks are used. All animals are observed for morbidity, mortality,injury, availability of food and water twice per day. Animals in poorhealth are euthanized. Blood samples are collected via cardiac punctureat each time point (0, 30 minutes, 2, 4, 8, 24 h). In addition, brainsare collected, and compound concentration determined at each time pointusing LCO2N MS/MS. Standard PK parameters are calculated for each routeof administration, including T1/2, Cmax, AUC, clearance, Vd and % F.

As an initial in vivo study, the effects of Fer-1 and SRS16-86 on serumcreatinine, a marker of kidney damage, were evaluated in an ischemiareperfusion injury (IRI) model of kidney failure (FIG. 21). Mice weredosed IP with each compound 2 mg/kg or with vehicle, 30 minutes prior toinduction of IRI. Both Fer-1 and SRS16-86 were effective in this simplein vivo test, which does not require substantial metabolic stability.However, more sophisticated in vivo models, such as the HD model,require optimized PK/PD parameters.

Example 17 Synchronized Renal Tubular Cell Death Involves Ferroptosis

Materials and Methods:

Mice

All WT mice (C57BL/6) reported in this study were obtained from CharlesRiver. Eight- to 12-wk-old male C57BL/6 mice (average weight˜23 g) wereused for all WT experiments, unless otherwise specified. Caspase-8 fl/flmice were kindly provided by Razquella Hakem (Department of MedicalBiophysics, University of Toronto, Toronto and Ontario Cancer Institute,University Health Network, Toronto). FADD fl/fl mice were generated byand provided by Manolis Pasparakis (Institute for Genetics, Universityof Cologne, Cologne, Germany). Doxycyclin-inducible renaltubule-specific Pax8-rtTA; Tet-on.Cre mice have been published (Galluzziet al., 2014) and were kindly provided by Tobias B. Huber (RenalDivision, University Medical Center Freiburg, Freiburg, Germany).RIPK3-deficient mice were kindly provided by Vishva M. Dixit. All invivo experiments were performed according to the Protection of AnimalsAct, after approval of the German local authorities or the InstitutionalAnimal Care and Use Committee (IA-CUC) of the University of Michigan,and the National Institutes of Health Guide for the Care and Use ofLaboratory Animals (National Research Counsel, 2011), after approvalfrom the University of Michigan IACUC or by the Local authoritiesresponsible for the approval at Ghent University. In all experiments,mice were carefully matched for age, sex, weight, and geneticbackground. Genotypes were confirmed by tail-snip PCR using thefollowing primers: Pax8-rtTA forward, 5′-CCATGTCTAGACTGGACAAGA-3′ (SEQID NO:8); Pax8-rtTA reverse, 5′-CTCCAGGCCACATATGATTAG-3′ (SEQ ID NO:9);tetO-Cre forward, 5′-GCATTACCGGTCGATGCAACGAGTGATGAG-3′ (SEQ ID NO:10);tetO-Cre reverse, 5′-GAGTGAACGAACCTGGTCGAAATCAGTGCG-3′ (SEQ ID NO:11);caspase-8 flox/flox forward, 5′-ATAATTCCCCCAAATCCTCGCATC-3′ (SEQ IDNO:12); caspase-8 flox/flox reverse, 5′-GGCTCACTCCCAGGGCTTCCT-3′ (SEQ IDNO:13); FADD flox/flox forward, 5′-TCACCGTTGCTCTTTGTCTAC-3′ (SEQ IDNO:14); FADD flox/flox reverse (I), 5′-GTAATCTCTGTAGGGAGCCCT-3′ (SEQ IDNO:15); and FADD flox/flox reverse (II), 5′-CTAGCGCATAGGATGATCAGA-3′(SEQ ID NO:16).

Histology, Immunohistochemistry, and Evaluation of Structural OrganDamage.

Organs were dissected as indicated in each experiment and infused with4% (vol/vol) neutral-buffered formaldehyde, fixated for 48 h, dehydratedin a graded ethanol series and xylene, and finally embedded in paraffin.Stained sections were analyzed using an Axio Imager microscope (Zeiss).Kidney damage was quantified by two experienced pathologists in adouble-blind manner on a scale ranging from 0 (unaffected tissue) to 10(severe organ damage). The following parameters were chosen asindicative of morphological damage to the kidney afterischemia-reperfusion injury (IRI): brush border loss, red blood cellextravasation, tubule dilatation, tubule degeneration, tubule necrosis,and tubular cast formation. These parameters were evaluated on a scaleof 0-10, which ranged from not present (0), mild (1-4), moderate (5 or6), severe (7 or 8), to very severe (9 or 10). Each parameter wasdetermined on at least four different animals.

Statistics.

For all experiments, differences of datasets were consideredstatistically significant when P values were lower than 0.05, if nototherwise specified. Statistical comparisons were performed using thetwo tailed Student t test. Asterisks are used in the figures to specifystatistical significance (*P<0.05; **P<0.02; ***P<0.001). P values insurvival experiments (Kaplan-Meier plots) were calculated using GraphPadPrism, ver. 5.04 software. Statistics are indicated as SD unlessotherwise specified.

Cell Lines and Reagents.

Necrostatin (Nec-1) was obtained from Sigma-Aldrich. Sanglifehrin A(SfA) was provided by Novartis Pharma. The zVAD-fmk (herein referred toas zVAD) was purchased from BD Biosciences. The monoclonal anti-Fasantibody was from Immunotech. Smac mimetics (Birinapant) was fromAbsource Diagnostics (Selleckchem). Murine NIH 3T3 fibroblasts wereoriginally obtained from ATCC and were cultured in Dulbecco's modifiedEagle medium (DMEM) (Invitrogen) supplemented with 10% (vol/vol) FCS,100 U/mL penicillin, and 100 μg/mL streptomycin. Human HT-1080fibrosarcoma cells were originally obtained from ATCC and were culturedin DMEM (Invitrogen) supplemented with MEM NEAA (Invitrogen), 10%(vol/vol) FCS, 100 U/mL penicillin, and 100 μg/mL streptomycin. MurineNIH 3T3 cells were originally obtained from ATCC and were cultured inDMEM (Invitrogen) supplemented with 10% (vol/vol) FCS, 100 U/mLpenicillin, and 100 μg/mL streptomycin. Jurkat cells were originallyobtained from ATCC and were cultured in RPMI 1640 supplemented with 10%(vol/vol) FCS, 100 U/mL penicillin, and 100 μg/mL streptomycin. All celllines were cultured in a humidified 5% CO₂ atmosphere.

Induction of Cell Death.

For induction of necroptosis, HT-29 cells were stimulated for 24 h at37° C. with 100 ng/mL TNFα plus 1 μM Smac mimetics plus 25 μM zVAD asindicated (vehicle-treated cells served as control). For induction offerroptosis, NIH and HT-1080 cells were each stimulated for 24 h at 37°C. with 50 μM erastin as indicated (vehicle-treated cells served as acontrol). For induction of apoptosis, Jurkat cells were stimulated for 4h with 100 ng/mL monoclonal anti-Fas (clone 7C11; Immunotech).Anti-Fas-induced apoptosis of Jurkat cells was inhibited by furtheraddition of 25 μM zVAD (vehicle-treated cells served as control).

Analysis of Cell Death.

For immunoblotting, cells were lysed in ice cold 10 mM Tris.HCl, pH7.5,50 mM NaCl, 1% Triton X-100, 30 mM sodium pyrophosphate, 50 mM NaF, 100μM Na₃VO₄, 2 μM ZnCl₂, and 1 mM phenylmethylsulfonyl fluoride (modifiedFrackelton buffer). Insoluble material was removed by centrifugation(14,000×g, 10 min, 4° C.), and protein concentration was determinedusing a commercial Bradford assay kit according to the manufacturer'sinstructions (Bio-Rad).

Equal amounts of protein (17 μg per lane) were resolved on a 12%SDS/PAGE gel and transferred to a nitrocellulose membrane (AmershamBiosciences). Western blot was performed using a polyclonal cleavedcaspase-3 antibody (Asp-175 from Cell Signaling) and a correspondingsecondary horseradish peroxidase linked polyclonal anti-rabbit antibody(Acris). Immune complexes were visualized by enhanced chemiluminescence(ECL; Amersham Biosciences).

Fluorescence-Activated Cell Sorting.

Phosphatidylserine exposure to the outer cell membrane of apoptoticcells or at the inner plasma membrane of necrotic cells andincorporation of 7-AAD into necrotic cells were quantified byfluorescence-activated cell sorting (FACS) analysis. The ApoAlertannexin V-FITC antibody and the 7-AAD antibody were purchased from BDBiosciences.

Isolation of Renal Tubules.

Six to 12 mice were used for each isolated tubule preparation, dependingon the amount of material needed for particular experiments. Forpreparation of isolated tubules, mice were anesthetized with ketamine(100 mg/kg i.p.) and xylazine (10 mg/kg i.p.), and the kidneys wereimmediately removed. Type I collagenase was from WorthingtonBiochemical. Percoll was purchased from Amersham Biosciences. All otherreagents and chemicals, including delipidated BSA, were of the highestgrade available from Sigma-Aldrich. Immediately after removal of thekidneys, the parenchyma was injected with 0.3-0.5 cc of a cold 95% O₂/5%CO₂-gassed solution consisting of 115 mM NaCl, 2.1 mM KCl, 25 mM NaHCO₃,1.2 mM KH₂PO₄, 2.5 mM CaCl₂, 1.2 mM MgCl₂, 1.2 mM MgSO₄, 25 mM mannitol,2.5 mg/mL fatty acid-free BSA, 5 mM glucose, 4 mM sodium lactate, 1 mMalanine, and 1 mM sodium butyrate (solution A) with the addition of 1mg/mL collagenase (type I; Worthington Biochemical). The cortices werethen dissected and minced on an ice-cold tile and then resuspended inadditional solution A for 8-10 min of digestion at 37° C., followed byenrichment of proximal tubules using centrifugation on self-formingPercoll gradients. For Nec-1 studies in renal tubules, renal corticeswere dissected in ice-cold dissection solution (DS) [HBSS with 10 mmol/Lglucose, 5 mmol/L glycine, 1 mmol/L alanine, 15 mmol/L Hepes (pH 7.4);osmolality, 325 mOsmol/L] and sliced into 1-mm pieces. The fragmentswere transferred to collagenase solution (DS with 0.1% (wt/vol) type 2collagenase and 96 μg/mL soybean trypsin inhibitor) and digested for 30min at 37° C. and 61×g. After digestion, the supernatant was sievedthrough two nylon sieves: first 250-μm pore size and then 100-μm poresize. The longer proximal tubule segments remaining in the 100-μm sievewere resuspended by flushing the sieve in the reverse direction withwarm DS (37° C.) containing BSA 1% (wt/vol). The proximal tubulesuspension was centrifuged for 5 min at 170×g, washed, and thenresuspended into the appropriate amount of culture medium (1:1 DMEM/F12without phenol red and supplemented with heat-inactivated 1% FCS, 15mmol/L Hepes, 2 mmol/L L-glutamine, 50 nmol/L hydrocortisone, 5 μg/mLinsulin, 5 μg/mL transferrin, 5 ng/mL sodium selenite, 0.55 mmol/Lsodium pyruvate, 10 mL/L 100× nonessential amino acids, 100 IU/mLpenicillin, and 100 μg/mL streptomycin buffered to pH 7.4 (osmolality of325 mOsmol/kg H₂O). The proximal tubule fragments were seeded onto atissue culture plate and cultured at 37° C. and 95% air/5% CO₂ in astandard humidified incubator.

In Vivo Microscopy on the Postischemic Cremaster Muscle.

The surgical preparation of the cremaster muscle was performed asoriginally described by Baez with minor modifications (Traykova-Brauchet al., 2008; Mulay et al., 2013). Mice were anesthetized using aketamine/xylazine mixture (100 mg/kg ketamine and 10 mg/kg xylazine),administrated by i.p. injection. The left femoral artery was cannulatedin a retrograde manner for administration of microspheres and drugs. Theright cremaster muscle was exposed through a ventral incision of thescrotum. The muscle was opened ventrally in a relatively avascular zone,using careful electrocautery to stop any bleeding, and spread over thetransparent pedestal of a custom-made microscopic stage. Epididymis andtesticle were detached from the cremaster muscle and placed into theabdominal cavity. Throughout the procedure as well as after surgicalpreparation during in vivo microscopy, the muscle was superfused withwarm-buffered saline.

The setup for in vivo microscopy was centered around equipment forstroboscopic fluorescence epiillumination microscopy. Light from a 75-Wxenon source was narrowed to a near monochromatic beam of a wavelengthof 700 nm by a galvanometric scanner (Polychrome II; TILL Photonics) anddirected onto the specimen via an FITC filter cube equipped withdichroic and emission filters (DCLP 500, LP515; Olympus). Microscopyimages were obtained with Olympus water immersion lenses (20×/numericalaperture (N.A.) 0.5 and 10×/N.A. 0.3) and recorded with an analogblack-and-white charge-coupled device (CCD) video camera (Cohu 4920;Cohu) and an analog video recorder (AG-7350-E; Panasonic). Obliqueillumination was obtained by positioning a mirroring surface (reflector)directly below the specimen and tilting its angle relative to thehorizontal plane. The reflector consisted of a round cover glass(thickness, 0.19-0.22 mm; diameter, 11.8 mm), which was coated withaluminum vapor (Freichel) and brought into direct contact with theoverlying specimen as described previously (Mulay et al., 2013). Formeasurement of centerline blood flow velocity, green fluorescentmicrospheres (2-μm diameter; Molecular Probes) were injected via thefemoral artery catheter, and their passage through the vessels ofinterest was recorded using the FITC filter cube under appropriatestroboscopic illumination (exposure, 1 ms; cycle time, 10 ms; length ofthe electromagnetic wave, 488 nm), integrating video images forsufficient time (>80 ms) to allow for the recording of several images ofthe same bead on one frame. Beads that were flowing freely along thecenterline of the vessels were used to determine blood-flow velocity.

For off-line analysis of parameters describing the sequential steps ofleukocyte extravasation, Cap-Image image analysis software (Dr. ZeintlBiomedical Engineering) was used. Rolling leukocytes were defined asthose moving slower than the associated blood flow and were quantifiedas described previously (Mulay et al., 2013). Firmly adherent cells weredetermined as those resting in the associated blood flow for more than30 s and related to the luminal surface per 100-μm vessel length.Transmigrated cells were counted in regions of interest (ROI), covering75 μm on both sides of a vessel over a 100-μm vessel length. Bymeasuring the distance between several images of one fluorescent beadunder stroboscopic illumination, centerline blood-flow velocity wasdetermined. From measured vessel diameters and centerline bloodflowvelocity, apparent wall-shear rates were calculated, assuming aparabolic flow-velocity profile over the vessel cross-section.

Animals were treated with ferrostatin-1 (2 mg/kg i.p.) or vehicle 30 minbefore the experiment. For the analysis of postischemic leukocyteresponses, three postcapillary vessel segments in a central area of thespread-out cremaster muscle were randomly chosen. After having obtainedbaseline recordings of leukocyte rolling, firm adhesion, andtransmigration in all three vessel segments, ischemia was induced byclamping all supplying vessels at the base of the cremaster muscle usinga vascular clamp (Martin). After 30 min of ischemia, the vascular clampwas removed, and reperfusion was restored for 160 min. Measurements wererepeated at 60 min and 120 min after onset of reperfusion. Subsequently,FITC dextran was infused intraarterial (i.a.) for the analysis ofmicrovascular permeability. After in vivo microscopy, blood samples werecollected by cardiac puncture for the determination of systemicleukocyte counts using a Coulter AcT Counter (Coulter). Anesthetizedanimals were then euthanized by exsanguination.

Analysis of microvascular permeability was performed as describedpreviously (Linkerman et al., 2012). Briefly, the macromoleculeFITC-dextran (5 mg in 0.1 mL saline, Mr 150,000; Sigma-Aldrich) wasinfused intraarterially after determination of centerline blood-flowvelocity. Five postcapillary vessel segments, as well as the surroundingperivascular tissue, were excited at 488 nm, and emission >515 nm wasrecorded by a CCD camera (Sensicam; PCO) 30 min after injection ofFITC-dextran using an appropriate emission filter (LP 515). Mean grayvalues of fluorescence intensity were measured by digital image analysis(TILLvisION 4.0; TILL Photonics) in six randomly selected ROIs (50×50μm2), localized 50 μm distant from the postcapillary venule underinvestigation. The average of mean gray values was calculated.

Acute Oxalate Nephropathy Model.

Mice were divided into two groups. One group received vehicle injectionsand another group received Ferrostatin-1 (10 mg/kg i.p.). Then, 15 minlater, both groups were given a single i.p. injection of 100 mg/kgsodium oxalate and 3% sodium oxalate in drinking water. Blood samplesand kidneys were harvested after 24 h. Kidneys were kept at −80° C. forprotein isolation and at −20° C. for RNA isolation. One part of thekidney was also kept in formalin to be embedded in paraffin forhistological analysis. Kidney sections (2 μm) were stained with periodicacid-Schiff (PAS) reagent. Tubular injury was scored by assessing thepercentage of necrotic tubules. Ly6B.2+ neutrophils were identified byimmunostaining (clone 7/4; Serotec). Pizzolato stain was used tovisualize CaOx crystals, and crystal deposit formation in the kidney wasevaluated as described previously (Mulay et al., 2013), using thefollowing point values: 0, no deposits; 1, crystals in papillary tip; 2,crystals in cortical medullary junction; 3, crystals in cortex. Whencrystals were observed in multiple areas, points were combined. Serumcreatinine and blood urea nitrogen were measured using Creatinine FS kitand Urea FS kit (both from DiaSys Diagnostic Systems) according to themanufacturer's protocol. Neutrophil infiltrates were counted in 15high-power fields (hpfs) per section. For real-time quantitative RT-PCR,total RNA was isolated from kidneys using a Qiagen RNA extraction kitfollowing the manufacturer's instructions. After quantification, RNAquality was assessed using agarose gels. From isolated RNA, cDNA wasprepared using reverse transcriptase (Superscript II; Invitrogen).Real-time quantitative RT-PCR (TaqMan) was performed using SYBR-GreenPCR master mix and analyzed with a Light Cycler 480 (Roche). All geneexpression values were normalized using 18S RNA as a housekeeping gene.All primers used for amplification were from Metabion. The expression ofKIM-1, IL-6, CXCL-2, and NF-κB p65 was analyzed using the followingprimers: Kim-1 forward, 5′-TCAGCTCGGGAATGCACA-3′ (SEQ ID NO:17); Kim-1reverse, 5′-TGGTTGCCTTCCGTGTCT-3′ (SEQ ID NO:18); IL-6 forward,5′-TGATGCACTTGCAGAAAACA-3′ (SEQ ID NO:19); IL-6 reverse,5′-ACCAGAGGAAATTTTCAATAG-3′ (SEQ ID NO:20); CXCL-2 forward,5′-TCCAGGTCAGTTAGCCTTGC-3′ (SEQ ID NO:21); CXCL-2 reverse,5′-CGGTCAAAAAGTTTGCCTTG-3′ (SEQ ID NO:22); NF-κB p65 forward,5′-CGCTTCTCTTCAATCCGGT-3′ (SEQ ID NO:23); NF-κB p65 reverse,5′-GAGTCTCCATGCAGCTACGG-3′ (SEQ ID NO:24).

Kidney Model of Ischemia-Reperfusion Injury.

Induction of kidney IRI was performed via a midline abdominal incisionand a bilateral renal pedicle clamping for either 40 min (severe IRI, or“lethal-to-WT” IRI) or 50 min (ultra-severe IRI) using microaneurysmclamps (Aesculab). Throughout the surgical procedure, the bodytemperature was maintained between 36° C. and 37° C. by continuousmonitoring using a temperature-controlled self-regulated heating system(Fine Science Tools). After removal of the clamps, reperfusion of thekidneys was confirmed visually. The abdomen was closed in two layersusing standard 6-0 sutures. Sham-operated mice underwent the identicalsurgical procedures, except that microaneurysm clamps were not applied.To maintain fluid balance, all of the mice were supplemented with 1 mLof prewarmed PBS administered intraperitoneally directly after surgery.All mice were killed 48 h after reperfusion for each experiment. Forassays investigating combination therapy with [Nec-1+SfA], mice receiveda total volume of 150 μL of Clinoleic with 10 mg of SfA per kg of bodyweight and 1.65 mg of Nec-1 per kg body weight 60 min before ischemia.Then, 2 mg of SRS16-86 or SRS16-79 per kg body weight were dissolved in2% DMSO and were applied intraperitoneally as a total volume of 400 μL15 min before the onset of surgery in all experiments. Allischemic-reperfusion experiments were performed in a double-blindedmanner.

Model of Cerulein-Induced Pancreatitis.

Cerulein-induced pancreatitis (CIP) has been described elsewhere indetail (3). Briefly, male C57BL/6 WT mice (n=7) and male RIPK3-ko mice(n=7) received i.p. injections of 50 μg cerulein/kg body weight onceevery hour for 10 h (in each case, 200 μL of total volume supplementedwith PBS). Animals were euthanized 24 h after the first injection, andsamples of blood and tissues were rapidly harvested. Quantification ofpancreas injury was performed by measuring serum amylase and lipaseactivity.

LPS-Induced Lethal Shock.

Mice were injected intraperitoneally (i.p.) with 15 mg/kg Escherichiacoli 0111:B4 LPS (Sigma-Aldrich). Mice were pretreated by an i.p.injection of 5 mg/kg ethyl 3-amino-4-(cyclohexylamino)benzoate (Fer-1;Matrix Scientific), 10 mg/kg[5-((7-Cl-1H-indol-3-yl)methyl)-3-methylimidazolidine2,4-dione] (Nec-1s;synthesized in house), a combination of Fer-1 and Nec-1s, or vehicle (1%DMSO in PBS) 30 min before the LPS challenge. Rectal body temperatureand survival rates were monitored daily up to 96 h. Experiments wereapproved by the animal ethics committee of Ghent University.

Results:

Conditional Deletion of FADD or Caspase-8 does not Induce Cell Death inRenal Tubular Epithelia.

The mode of cell death of tubular cells in acute kidney injury (AKI) hasbeen a matter of intense discussion (Linkerman et al., 2012; Linkermanet a., 2013A) Because RIPK3-deficient mice have been shown to bepartially protected from IRI-induced tubular necrosis (Linkerman et al.,2013B; Luedde et al., 2014) and because Nec-1 phenocopies this effect(Linkerman et al., 2012; Degterev et al., 2005; Smith et al., 2007), itwas hypothesized that necroptosis might be the mode of cell death thatdrives parenchymal cells into necrosis. However, it was not ruled outthat tubular cell death might have occurred secondary to some changesoutside the tubular compartment: e.g., in RIPK3-dependent organperfusion, which might be altered also upon Nec-1 treatment if thenecroptotic pathway was involved. In fact, a discrepancy between thestrong in vivo protection and the marginal protective effect ofRIPK3-deficient freshly isolated tubules would be consistent with thishypothesis (Linkerman et al., 2013B). A powerful approach todefinitively study the involvement of cell-specific necroptosis is todelete components of the necroptosis-suppressing complex, which consistsof FADD, caspase-8, RIPK1, and FLIP (Dillon et al., 2012; Dillon et al.,2014), and to analyze spontaneously occurring necroptosis. Therefore,tubular cell-specific inducible conditional mice (Pax8-rtTA Tet-on.Cre)(Traykova-Brauch et al., 2008) were crossed to FADD or capase-8 floxedmice (FIG. 22 A-E) and confirmed the deficiency of the protein ofinterest by Western blotting from freshly isolated renal tubules (FIGS.23 A and B and D and E), which were carefully confirmed to be devoid ofany other cells (FIG. 24). Unlike in all other tissues reported so far,inducible deletion of FADD or caspase-8 in Pax8-rtTA; Tet-on.Cre×FADDfl/fl and Pax8-rtTA; Tet-on.Cre×caspase-8 fl/fl mice did not affectserum markers of renal function (FIG. 23G and FIG. 22F), histologicalappearance (FIGS. 23 C and F), or organ function for the entireobservation period of 3 wk after addition of doxycycline into thedrinking water (FIG. 23G and FIG. 22F). After the 3-wk period ofdoxycyclin feeding, mice were morphologically indistinguishable fromnondoxy-fed littermates (FIG. 22E). In addition, induction of acutekidney injury by application of the nephrotoxin cisplatin (20 mg/kg bodyweight) led to similar survival kinetics as in WT mice (FIG. 23H). Infreshly isolated renal tubules treated with 50 μM Nec-1, no protectionwas detected both in the presence or in the absence of glycine (FIG.23I). This result is in line with our previous observation of low levelsof RIPK3 in tubular cell lines (Linkerman et al., 2012) and marginalsensitivity for necroptosis compared with a glomerular endothelial cellline and L929 fibrosarcoma cells (Linkerman et aL, 2012). Takentogether, these data strongly argue against necroptosis as the primarymode of regulated cell death in renal tubules and suggest that theeffects in RIPK3-ko and Nec-1-treated mice are due to extratubulareffects.

RIPK3-Deficient Mice Exhibit Increased Renal Perfusion and Fail to GainNormal Body Weight.

Using low-resolution intravital microscopy, the effects of Nec-1 on thediameter of peritubular capillaries were previously investigated(Linkerman et aL, 2013A). Using a similar approach with higherresolution (FIG. 25A), RIPK3-ko mice were analyzed and statisticallysignificant increases in peritubular diameters were detected (FIGS. 25 Band C), which would account for 25.7% increase in blood flow (FIG. 25D)according to the law of Hagen Poiseuille and could help maintainperitubular perfusion after ischemia when it is known to be compromised.Taking into consideration that, in all renal cells investigated, thehighest RIPK3 expression was found in endothelial cells, vasculareffects cannot be ruled out, and endothelial cell-specific conditionalRIPK3 depletion should be investigated in IRI models. In this respect,it is noteworthy that, in another model of necrotic parenchymal damagethat does not rely on ischemia-reperfusion injury, the cerulein-inducedpancreatitis, in our hands, RIPK3-deficient mice are not protected fromthe increase in serum levels of amylase and lipase (FIG. 25E). Inaddition, RIPK3-ko mice fail to gain normal body weight in comparisonwith WT littermates in both males and females (FIGS. 25 F and G).

Morphological Changes of Synchronized Tubular Cell Death are Preventedby Ferrostatins.

If apoptosis and necroptosis account for only minor damage of acutetubular necrosis, then what pathway of regulated necrosis might becausative of synchronized tubular death that was identified in animproved ex vivo model of freshly isolated murine renal tubules upondepletion of fatty acids (FIG. 26A). It was confirmed that such tubulesare functional before the onset of the fatty-acid depletion for maximalcontrol purposes. Importantly, such synchronized tubular (cell) deathvery much resembles the appearance of casts found in urine sediments ofpatients with acute kidney injury. The dynamics of the tubular necrosissuggest a direct cell-to-cell communication to deliver the deadlysignal. Because a beneficial effect of the second generation ferrostatin11-92 (Skouta et al., 2014) in a model of acute injury of freshlyisolated renal tubules was previously reported, the morphology of suchtubules in the presence of erastin, a well-described inducer offerroptosis, a necrotic type cell death that largely depends on lipidperoxidation (Dixon et al., 2012, Yagoda et al., 2014), over time (FIG.26B). Whereas the faint plasma membrane of untreated tubules did notchange in SRS16-86-treated tubules (a novel third-generation ferrostatinwas looked for (FIG. 27)), vehicle-treated and especiallyerastin-treated tubules showed membrane protrusions and obvious signs ofdeformation and functional insufficiency (FIG. 26B). When erastin wasapplied into the tubule, a similar type of cell death occurred evenwithout fatty-acid depletion. In another well established lactatedehydrogenase (LDH) release-based assay of ex vivo tubulotoxicity(Skouta et al., 2014), hydroxyquinoline-iron-induced cell death, Fer-1and the Nox-inhibitor GKT were found to be protective whereas Nec-1 didnot show any protection (FIG. 26C).

Ferroptosis Mediates IRI-Mediated Immune-Cell Infiltration of theCremaster Muscle.

The concept of immunogenic necrotic cell death proposes to trigger anautoamplification loop, which is triggered by a necroinflammatorymicroenvironment. To understand how ferroptosis attracts immune cellsduring hypoxia/reperfusion situations, musculus cremaster IRI assays andread out the immune-cell infiltration by intravital microscopy (FIG. 26D-F) and quantified rolling, adherent, and transmigrated cells in thepresence and absence of ferrostatins were performed (FIGS. 26 F and Gand FIG. 28). Less immune-cell infiltration into the postischemic areasuggested either that Fer-1 directly inhibits leukocyte transmigrationor that the local proinflammatory microenvironment was lesschemoattractive, presumably by less damage-associated molecular pattern(DAMP) release due to less ferroptotic cell death.

Table 5 shows the results of a measurement of microhemodynamicparameters and systemic leukocyte counts, as indicated, based onintravital microscopy in the presence and absence of Fer-1.

TABLE 5 Leukocyte adherence upon addition of Fer-1 Vehicle FerrostatinInner diameter (μm) 27.6 ± 2.2  25.9 ± 1.6  Vmean (mm⁻¹ s⁻¹) 0.8 ± 0.10.8 ± 0.2 Shear rate (s⁻¹) 1208.8 ± 257.4  1272.0 ± 167.3  SLC (n ×10⁻⁸/μl 3.1 ± 3.6 1.6 ± 1.5Ferroptosis Mediates Tubule Necrosis in Oxalate Nephropathy, but not inan LPS-Induced Septic Shock Model.

Having identified ferroptosis to be of relevance in acute postischemicinjury, another model of acute renal failure, oxalate nephropathy, whichhas recently been established was investigated (Mulay et al., 2013).Intrarenal CaOx crystal deposition was identical in all groups (FIGS. 29A and B). However, neutrophil infiltration and expression levels ofproinflammatory cytokines (CXCL-2, IL-6), kidney injury molecule 1(KIM-1), and the p65 subunit of NF-κB were statistically significantlyreduced upon a once daily i.p. application of Fer-1 (FIG. 29E).Importantly, Fer-1 also significantly reduced the tubular injury score(FIG. 29D) and the functional serum markers of kidney injury creatinineand urea (FIG. 29C). Because RIPK3-deficient mice have been demonstratedto be resistant to sepsis models, Fer-1 in the model of LPS-inducedshock was also investigated (FIG. 29F), but no difference compared withvehicle-treated mice was evident. Therefore, ferroptosis seems to be avaluable target for both postischemic and toxic acute kidney injury.However, given the poor plasma stability of Fer-1, a more stablecompound for the in vivo applications was developed.

Ferroptosis Mediates Necrotic Tubular Cell Death in RenalIschemia—Reperfusion Injury.

For in vivo experiments, SRS16-86 was used in comparison with SRS16-79,an inactive derivative (FIG. 27B). The ferroptosis-inhibiting ability ofSRS16-86 and SRS16-79 was confirmed in vitro by FACS analysis oferastin-treated HT1080 cells and in NIH 3T3 cells (FIG. 27C). In bothnecroptosis and ferroptosis, cleaved caspase 3 was not detected (FIG.27D).

Active (SRS16-86) and inactive (SRS16-79) compounds were applied to amodel of severe ischemia-reperfusion injury (IRI), which was describedpreviously (Linkerman et al., 2013B). Upon 40 min of ischemia beforereperfusion, it is known that all WT mice die between 48 h and 72 h,which can be anticipated from the 48-h serum creatinine values above 2.0mg/dL. As demonstrated in FIG. 30 A-D, creatinine levels of allvehicle-treated C57BL/6 mice exceeded 2.0 mg/dL and showed strongmorphologic damage and high serum urea levels whereas Fer-1-treated and,to a greater extent, SRS16-86-treated mice were protected fromfunctional acute renal failure (FIGS. 30 C and D) and structural organdamage (FIGS. 30 A and B) after IRI. Mice treated with SRS16-79 in asimilar manner did not show any differences compared with thevehicle-treated controls. Side effects from treatment with the same doseof SRS16-86 4 wk after application were not observed. The effect ofSRS16-86 exceeded that of any single compound previously experienced tobe protective from renal IRI.

Ferrostatins Further Increase the Protective Effect ofNecrostatin-1/Sanglifehrin A Combination Therapy in Renal IRI.

Given that Nec-1 protects from renal IRI to a lesser extent thanSRS16-86, and given that interference with mitochondrial permeabilitytransition (MPT)-induced regulated necrosis (MPT-RN) by the compoundsanglifehrin A (SfA) also mildly protects from IRI, with marked additiveprotective effects in combination therapy with Nec-1 (Linkerman et al.,2013B; Linkerman et al., 2014A), the influence of SRS16-86 and SRS16-79in Nec-1+SfA-treated mice was investigated. Because the effect of theNec-1+SfA treatment could be investigated only in a severe ischemiamodel and additive protective effects by SRS16-86 were underinvestigation, the ischemic duration was increased to create a model ofultrasevere IRI (see Materials and Methods for details). In suchsettings, even the combination therapy with Nec-1+SfA did not fullyrescue creatinine values and organ damage (FIG. 30 E-H). Addition ofSRS16-86, but not SRS16-79, reduced plasma levels of serum urea andserum creatinine, suggesting that a triple combination therapy withNec-1+SfA plus SRS16-86 is superior in the prevention of renal IRIcompared with the double-combination therapy with Nec-1+SfA. Inaddition, this result indicates either that at least three independentpathways of regulated necrosis may be involved in IR-mediated organdamage or that inhibition of overlapping elements with SfA and Nec-1 areincomplete.

Discussion

Parenchymal cell necrosis, but not apoptosis, which seems to beminimally involved in the pathogenesis of acute kidney injury (Linkermanet al., 2014), triggers the release of DAMPs, some of which can triggerregulated necrosis and therefore initiate a necroinflammatory feedbackloop. In clinical routine, immunosuppression is a standard procedure,but an anti-cell death therapy does not exist apart from cyclosporine(Linkerman et al., 2014A). Therefore, defining the precise mechanismsthat trigger regulated necrosis is essential for the development of newdrugs. Our data indicate that alternative interpretations apart from“pure” necroptosis exist and suggest that ferroptosis is of importancein renal tubules, which, other than the skin, the gastrointestinaltract, or immune cells, do not depend on a necroptosis-inhibitingcomplex, at least not of FADD and caspase-8. To date, it cannot be ruledout that previously described protection against ischemia—reperfusioninjury in diverse organs is mediated by increased perfusion rather thandirect protection from parenchymal necroptosis. Our results regardingthe cerulein-induced pancreatitis are in line with this hypothesis andare in contrast to previously published data (He et al., 2007; Zhang etal., 2009). Several unanswered questions remain: Why are RIPK3-deficientmice protected in several models of IRI if the parenchymal cells do notundergo necroptosis? Why do necrostatins protect such tissues from organfailure if necroptosis is not the predominant mechanism of parenchymalcell death? Endothelial cell-specific deletion of RIPK3 will help toanswer these open questions.

DOCUMENTS

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All documents cited in this application are hereby incorporated byreference as if recited in full herein.

Although illustrative embodiments of the present invention have beendescribed herein, it should be understood that the invention is notlimited to those described, and that various other changes ormodifications may be made by one skilled in the art without departingfrom the scope or spirit of the invention.

What is claimed is:
 1. A compound according to formula (I):

wherein: R₁ and R₂, are independently selected from the group consistingof no atom, H, D, ═O, —O⁻, halo, C₁₋₆alkyl, C₁₋₆alkyl-aryl,C₁₋₆alkyl-heteroaryl, C₂₋₆alkenyl, C₂₋₆alkenyl-aryl, andC₂₋₆alkenyl-heteroaryl, wherein the C₁₋₆alkyl, C₁₋₆alkyl-aryl,C₁₋₆alkyl-heteroaryl, C₂₋₆alkenyl, C₂₋₆alkenyl-aryl, andC₂₋₆alkenyl-heteroaryl may be optionally substituted with an atom or agroup selected from the group consisting of halo, deuterium, C₁₋₄alkyl,CF₃, and combinations thereof; and R₃ is independently selected from thegroup consisting of H, C₁₋₁₂aliphatic, C₁₋₆-alkyl-aryl andC₁₋₆-alkyl-heteroaryl; or an N-oxide, hydrate, or pharmaceuticallyacceptable salt thereof, with the proviso that: when R₃ is ethyl, R₁ andR₂ cannot be both H, ═O, —O⁻, or

 and when R₃ is ethyl and at least one of R₁ or R₂ is H, then the otherof R₁ or R₂ cannot be

R₁ and R₂ are not both “no atom”; and when R₃ is H, then at least one ofR₁ and R₂ is not H.
 2. A compound having the structure of formula (II):

wherein: R₃ is independently selected from the group consisting of H,C₁₋₁₂aliphatic, C₁₋₆-alkyl-aryl and C₁₋₆-alkyl-heteroaryl; R₄ and R₅ areindependently selected from the group consisting of no atom, H, D, ═O,—O⁻, halo, aryl, heteroaryl, C₁₋₆alkyl, C₁₋₆alkyl-aryl, andC₁₋₆alkyl-heteroaryl; and --- is an optional bond, or an N-oxide,hydrate, or pharmaceutically acceptable salt thereof.
 3. A compoundhaving the structure of formula (III):

wherein the C₂₋₄ alkyl is selected from the group consisting of ethyl,isopropyl, n-propyl, butyl, and t-butyl, or an N-oxide, hydrate, orpharmaceutically acceptable salt thereof.
 4. A compound selected fromthe group consisting of:

or an N-oxide, hydrate, or pharmaceutically acceptable salt thereof. 5.A compound according to claim 3, which is selected from the groupconsisting of:

and combinations thereof, or an N-oxide, hydrate, or pharmaceuticallyacceptable salt thereof.
 6. A pharmaceutical composition comprising apharmaceutically acceptable carrier or diluent and a compound accordingto formula (I):

wherein: R₁ and R₂, are independently selected from the group consistingof no atom, H, D, ═O, —O⁻, halo, C₁₋₆alkyl, C₁₋₆alkyl-aryl,C₁₋₆alkyl-heteroaryl, C₂₋₆alkenyl, C₂₋₆alkenyl-aryl, andC₂₋₆alkenyl-heteroaryl, wherein the C₁₋₆alkyl-aryl,C₁₋₆alkyl-heteroaryl, C₂₋₆alkenyl, C₂₋₆alkenyl-aryl, andC₂₋₆alkenyl-heteroaryl may be optionally substituted with an atom or agroup selected from the group consisting of halo, deuterium, C₁₋₄alkyl,CF₃, and combinations thereof; and R₃ is independently selected from thegroup consisting of H, C₁₋₁₂aliphatic, C₁₋₆-alkyl-aryl andC₁₋₆-alkyl-heteroaryl; or an N-oxide, hydrate, or pharmaceuticallyacceptable salt thereof, with the proviso that: when R₃ is ethyl, R₁ andR₂ cannot be both H, ═O, —O⁻,or

 and when R₃ is ethyl and at least one of R₁ or R₂ is H, then the otherof R₁ or R₂ cannot be

R₁ and R₂ are not both “no atom”; and when R₃ is H, then at least one ofR₁ and R₂ is not H.
 7. A pharmaceutical composition comprising apharmaceutically acceptable carrier or diluent and a compound accordingto formula (II):

wherein: R₃ is independently selected from the group consisting of H,C₁₋₁₂aliphatic, C₁₋₆-alkyl-aryl and C₁₋₆-alkyl-heteroaryl; R₄ and R₅ areindependently selected from the group consisting of no atom, H, D, ═O,—O⁻, halo, aryl, heteroaryl, C₁₋₆alkyl, C₁₋₆alkyl-aryl, andC₁₋₆alkyl-heteroaryl; and --- is an optional bond, or an N-oxide,hydrate, or pharmaceutically acceptable salt thereof.
 8. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier or diluent and a compound according to formula (III):

wherein the C₂₋₄ alkyl is selected from the group consisting of ethyl,isopropyl, n-propyl, butyl, and t-butyl, or an N-oxide, hydrate, orpharmaceutically acceptable salt thereof.
 9. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier or diluentand a compound having a structure that is selected from the groupconsisting of:

and combinations thereof, or an N-oxide, hydrate, or pharmaceuticallyacceptable salt thereof.
 10. A pharmaceutical composition according toclaim 8, wherein the compound has a structure that is selected from thegroup consisting of:

and combinations thereof or an N-oxide, hydrate, or pharmaceuticallyacceptable salt thereof.
 11. A kit comprising a compound according toclaim 3 together with instructions for the use of the compound.
 12. Akit comprising a pharmaceutical composition according to claim 8together with instructions for the use of the pharmaceuticalcomposition.
 13. A compound having the formula: